Composition and methods for preventing or reducing the incidence of transient ischemic attacks

ABSTRACT

A composition and method for preventing or reducing the incidence of a transient ischemic attack in a subject at risk for developing a stroke comprises orally administering to the subject a prophylactically effective amount of a pharmaceutical composition comprising an ASIC1a inhibitor capable of penetrating the blood-brain barrier. Preferred ASIC1a inhibitors for use in the disclosed methods include amiloride and amiloride analogs.

This application is a continuation application of U.S. patentapplication Ser. No. 16/791,055, filed Feb. 14, 2020, which is acontinuation application of U.S. patent application Ser. No. 16/114,815,filed Aug. 28, 2018, now U.S. Pat. No. 10,603,316, which is acontinuation-in-part application of U.S. patent application Ser. No.15/445,833, filed on Feb. 28, 2017, now U.S. Pat. No. 10,201,539, whichis a continuation application of U.S. patent application Ser. No.13/972,558, filed Aug. 21, 2013, now U.S. Pat. No. 9,629,838. Theentirety of all of the aforementioned applications is incorporatedherein by reference.

FIELD

This application relates to the field of neurology and cardiology. Inparticular, this application related to compositions comprising anASIC1a inhibitor for preventing or reducing the incidence of TIAs andnerve injuries in a subject.

BACKGROUND

A transient ischemic attack (“TIA” or “mini stroke”) is an acute episodeof temporary neurologic dysfunction, typically lasting less than an hour(sometimes up to 24 hours). A TIA is caused by a brief interruption ofoxygen flow or transient disturbance of blood supply to a tissue,typically due to an obstructing blood clot without infarction. Whensymptoms persist for a longer time and are accompanied by infarction,the dysfunction is categorized as a stroke.

Whereas the classical definition of TIA included symptoms lasting aslong as 24 h, advances in neuroimaging have suggested that many suchcases represent minor strokes with resolved symptoms rather than trueTIAs. Thus, the American Heart Association and the American StrokeAssociation (ASA) endorse a tissue-based definition of TIA (i.e., as anepisode of focal ischemia rather than acute infarction) rather than atime-based definition.

The most common cause of a TIA is an embolus (blood clot) that occludesan artery in the brain, and to a lesser extent, the spinal cord andretina. Typically, the clots result from an atherosclerotic plaque inone of the two carotid arteries, or from the heart, for example, inpatients with atrial fibrillation (AFIB). Symptoms of TIA typicallyinclude temporary amaurosis (loss of vision), aphasia (difficultyspeaking), hemiparesis (weakness of one side of the body, and/orparesthesia (numbness).

TIAs are often considered as a warning for an approaching stroke.Identification of TIAs is important as the incidence of a subsequentstroke is as high as 11% over the next 7 days and 24-29% over thefollowing 5 years. However, up to 80% of strokes following TIA arepreventable. Accordingly, early diagnosis and treatment are critical.

According to current guidelines of the American Stroke Association(ASA), risk factors include nonmodifiable factors (age, sex, race, andsignificant family history); modifiable factors (body weight,hypertension, unhealthy lipid profile, cerebral microbleeds,cardiovascular diseases, including coronary artery disease, myocardialinfarction, peripheral arterial disease, valvular disease, atrialfibrillation, atrial flutter; diabetes mellitus; and lifestyle choices(cigarette smoking, alcohol consumption, illicit drug use, unhealthydiet/poor nutrition, and physical inactivity).

Patients with atrial fibrillation (AFIB) and atrial flutter (AFL) are ata higher risk for developing TIAs and strokes. AFIB affects about 2.3million people in North America and 4.5 million people in the EuropeanUnion and is emerging as a growing public health concern because of theaging of the population. AFIB is a condition in which the upper chambersof the heart beat in an uncoordinated and disorganized fashion,resulting in a very irregular and fast rhythm (i.e., an irregularly,irregular heartbeat). When blood is not completely pumped out of theheart's chambers, it can pool and clot. If a blood clot forms in theatria, exits the heart and blocks an artery in the brain, a TIA orstroke results. Consequently, about 15 percent of strokes result fromAFIB.

AFL is a common abnormal heart rhythm, similar to AFIB, the most commonabnormal heart rhythm. Both conditions are types of supraventricular(above the ventricles) tachycardia (rapid heartbeat). In AFIB, the heartbeats fast and in no regular pattern or rhythm. By contrast, in AFL, theupper chambers (atria) of the heart beat abnormally fast, but in aregular pattern, resulting in atrial muscle contractions that are fasterthan and out of sync with the lower chambers (ventricles). AFL patientsexhibit a distinct “sawtooth” pattern on an electrocardiogram (ECG), atest used to diagnose abnormal heart rhythms.

If left untreated, the side effects of AFIB and AFL can be potentiallylife threatening. With blood pooling in the heart (AFIB) or moving moreslowly (AFL), it is more likely to form clots. If the clot is pumped outof the heart, it could travel to the brain, spinal cord, or retina,thereby causing a TIA or stroke.

TIAs and strokes represent different ends of an ischemic continuum fromthe physiological perspective, but clinical management is similar. Insome cases, antiplatelet drugs have been found to be effective inpreventing TIAs. Many physicians believe antithrombotic therapy shouldbe initiated as soon as intracranial hemorrhage has been ruled out. Forpatients with TIA or ischemic stroke of cardiac origin due to atrialfibrillation, vitamin K antagonists (VKAs) are highly effective inpreventing recurrent ischemic stroke but have important limitations andare consequently underused. Antiplatelet therapy is less effective thanVKAs. The direct thrombin inhibitor, dabigatran etexilate, has shownefficacy over warfarin in a recent trial. Other anticoagulants includeoral factor Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban;the parenteral factor Xa inhibitor, idrabiotaparinux, and the VKA,tecarfarin.

Notwithstanding the risks for strokes in patients with AFIB and AFL,however, strokes mainly occur in patients without AFIB or AFL. In viewof the foregoing, there is a need for improved medications forpreventing TIAs and nerve injuries in patients at risk.

SUMMARY

One aspect of the present application related to a method for preventingor reducing the incidence of a transient ischemic attack in a subject atrisk for developing a transient ischemic attack, comprising orallyadministering to the subject a prophylactically effective amount of apharmaceutical composition comprising an ASIC1a inhibitor capable ofpenetrating the blood-brain barrier.

In one embodiment, the ASIC1a inhibitor comprises amiloride, anamiloride analog, or a pharmaceutically acceptable salt or solvatethereof.

In a particular embodiment, the ASIC1a inhibitor comprises amiloride ora pharmaceutically acceptable salt or solvate thereof.

In another embodiment, the ASIC1a inhibitor comprises an amilorideanalog or a pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, the amiloride analog is selected from the groupconsisting of benzamil, bepridil, KB-R7943, phenamil, 5-(N—N-dimethyl)amiloride (DMA), 5-(N,N-hexamethylene) amiloride (HMA),5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N-methyl-N-isopropyl (MIA),pharmaceutically acceptable salts or solvates thereof, methylatedanalogs thereof, and combinations thereof.

In other embodiments, the amiloride analog is selected from the groupconsisting of methylated analogs of benzamil, amiloride analogscontaining a ring formed on a guanidine group, amiloride analogscontaining an acylguanidino group, and amiloride analogs containing awater solubilizing group formed on a guanidine group, wherein the watersolubilizing group is a N,N-dimethyl amino group or a sugar group.

In one embodiment, the pharmaceutical composition is administered daily.

In another embodiment, the pharmaceutical composition is formulated asan extended release formulation.

In preferred embodiments, the subject is at risk for developing TIAs orstrokes.

In one embodiment, the subject has recently had heart surgery or haspreviously had a TIA or stroke.

In another embodiment, subject has an abnormal heart rhythm selectedfrom the group consisting of atrial fibrillation, atrial flutter,ventricular tachycardia, and ventricular fibrillation.

In another embodiment, the subject has acute coronary syndrome, arterialembolism, atherosclerosis, atrial fibrillation, carotid artery disease,cerebral arterial thrombosis, cerebral embolism, coronary arterialthrombosis, coronary heart disease, deep vein thrombosis, kidneyembolism, myocardial infarction, peripheral arteriopathy, pulmonaryembolism, stroke, thrombophlebitis, thrombosis, transient ischemicattack, unstable angina, valvular heart disease, venous thrombosis,ventricular fibrillation, or a combination thereof.

In one embodiment, the amiloride, amiloride analog or a pharmaceuticallyacceptable salt or solvate thereof is administered in a dose range of0.1 mg-10 mg/kg body weight.

In another aspect, the pharmaceutical composition further includes oneor more anti-clotting agents, such as antiplatelet agents, anticoagulantagents, anti-arrhythmic agents, or a combination thereof.

In one embodiment, the pharmaceutical composition includes anantiplatelet agent selected from the group consisting of aspirin,clopidogrel, prasugrel, ticagrelor, dipyridamole, and combinationsthereof.

In another embodiment, the pharmaceutical composition includes ananticoagulant agent selected from the group consisting of vitamin-Kepoxide reductase inhibitors, direct thrombin inhibitors and Factor Xainhibitors. In a particular embodiment, the anticoagulant agent isselected from the group consisting of heparin, warfarin, dabigatran,apixaban, edoxaban, rivoraxaban, ximelagatran, argatroban, AZD-0837,YM466, betrixaban, edoxaban, tecarfarin, and combinations thereof.

In another embodiment, the pharmaceutical composition includes ananti-arrhythmic agent selected from the group consisting of dronedarone,budiodarone, amiodarone, vernakalant, celivarone, AZD-1305, dofetilide,ibutilide, flecainide, quinidine, sotolol, propafenone, and combinationsthereof.

In another aspect, a pharmaceutical composition for reducing nervoussystem injury, comprises an effective amount of one or more ASIC1ainhibitors selected from the group consisting of amiloride, amilorideanalogs, pharmaceutically acceptable salts or solvates thereof,methylated analogs thereof, and combinations thereof; and apharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition is formulated for oral administration of the one or moreASIC1a inhibitors, wherein the ASIC1a inhibitor(s) are capable ofpenetrating the blood-brain barrier.

In one embodiment, the ASIC1a inhibitor comprises amiloride or apharmaceutically acceptable salt or solvate thereof.

In another embodiment, the ASIC1a inhibitor comprises an amilorideanalog or a pharmaceutically acceptable salt or solvate thereof. In aparticular embodiment, the amiloride analog is selected from the groupconsisting of benzamil, bepridil, KB-R7943, phenamil, 5-(N—N-dimethyl)amiloride (DMA), 5-(N,N-hexamethylene) amiloride (HMA),5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N-methyl-N-isopropyl (MIA),pharmaceutically acceptable salts or solvates thereof, methylatedanalogs thereof, and combinations thereof.

In another embodiment, the amiloride analog is selected from the groupconsisting of methylated analogs of benzamil, amiloride analogscontaining a ring formed on a guanidine group, amiloride analogscontaining an acylguanidino group, and amiloride analogs containing awater solubilizing group formed on a guanidine group, wherein the watersolubilizing group is a NN-dimethyl amino group or a sugar group.

In another embodiment, the pharmaceutical composition comprises anextended release formulation for delivery of the one or more ASIC1ainhibitors.

In other embodiments, the pharmaceutical composition further comprisesone or more antiplatelet agents selected from the group consisting ofaspirin, clopidogrel, prasugrel, and ticagrelor, and combinationsthereof.

In another embodiment, the pharmaceutical composition further comprisesone or more anticoagulant agents selected from the group consisting ofvitamin-K epoxide reductase inhibitors, direct thrombin inhibitors andFactor Xa inhibitors. In certain embodiments, the one or moreanticoagulant agents are selected from the group consisting of apixaban,argatroban, AZD-0837, betrixaban, dabigatran, edoxaban, heparin,rivoraxaban, tecarfarin, warfarin, ximelagatran, YM466, and combinationsthereof.

In another embodiment, the pharmaceutical composition further comprisesone or more anti-arrhythmic agents. In certain embodiments, the moreanti-arrhythmic agents are selected from the group consisting ofamiodarone, AZD-1305, budiodarone, celivarone, dofetilide, dronedarone,flecainide, ibutilide, propafenone, quinidine, sotolol, vernakalant, andcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of a flowchart illustrating an exemplary method ofreducing neuroinjury in an ischemic subject.

FIG. 2 is a view of a flowchart illustrating an exemplary method ofidentifying drugs for treating ischemia-related nerve injury.

FIGS. 3A-D are a series of graphs presenting exemplary data related tothe electrophysiology and pharmacology of acid sensing ion channel(ASIC) proteins in cultured mouse cortical neurons.

FIGS. 4A-D are an additional series of graphs presenting exemplary datarelated to the electrophysiology and pharmacology of ASIC proteins incultured mouse cortical neurons.

FIGS. 5A-D are a set of graphs and traces presenting exemplary datashowing that modeled ischemia may enhance activity of ASIC proteins, inaccordance with aspects of the present teachings.

FIGS. 6A-B and 7A-D are a set of graphs and traces presenting exemplarydata showing that ASIC proteins in cortical neurons may be Ca²⁺permeable, and that Ca²⁺ permeability may be ASIC1a dependent.

FIGS. 8A-C are a series of graphs presenting exemplary data showing thatacid incubation may induce glutamate receptor-independent neuronalinjury that is protected by ASIC blockade.

FIGS. 9A-D are a series of graphs presenting exemplary data showing thatASIC1a may be involved in acid-induced injury in vitro.

FIGS. 10A-D are a series of graphs with data showing neuroprotection inbrain ischemia in vivo by ASIC1a blockade and by ASIC1 gene knockout.

FIG. 11 is a graph plotting exemplary data for the percentage ofischemic damage produced by stroke in an animal model system as afunction of the time and type of treatment.

FIG. 12 is a view of the primary amino acid sequence of an exemplarycystine knot peptide, PcTx1 (SEQ. ID. NO. 1), with various exemplarypeptide features shown.

FIG. 13 is a comparative view of the cystine knot peptide of FIG. 12aligned with various exemplary deletion derivatives of the peptide.

FIG. 14 is an exemplary graph plotting the amplitude of calcium currentmeasured in cells as a function of the ASIC family member(s) expressedin the cells.

FIG. 15 is a graph presenting exemplary data related to the efficacy ofnasally administered PcTx venom in reducing ischemic injury in an animalmodel system.

FIGS. 16A-C are a composite showing representative ASIC 1a currenttraces in CHO cells treated with benzamil (panel A) or5-(N-ethyl-N-isopropyl) amiloride (EIPA) (panel B), and dose-dependentblockade of ASIC 1a current expressed in CHO cells by amiloride andamiloride analogs (panel C).

FIGS. 17A-C are a composite showing representative ASIC 2a currenttraces in CHO cells treated with benzamil (panel A) or amiloride (panelB), and dose-dependent blockade of ASIC 2a current expressed in CHOcells by amiloride and amiloride analogs (panel C).

FIG. 18 is a graph showing reduction of infarct volume in mice byintracerebroventricular injections of amiloride or amiloride analogs.

FIG. 19 is a composite showing reduction of infarct volume in thecortical tissue of mice by intravenous injection of saline or amiloride60 min after MCAO.

FIG. 20 is a composite showing reduction of infarct volume in thecortical tissue of mice by intravenous injection of saline or amiloride3 hours or 5 hours after MCAO.

FIG. 21 shows structure activity relationship (SAR) for hydrophobicamiloride analogs on various channels.

DETAILED DESCRIPTION Definitions

As used herein, the term “nervous system” includes both the centralnervous system and the peripheral nervous system.

The term “amiloride analog” includes structural analogs of amiloride,functional analogs of amiloride, or a combination thereof.

The term “central nervous system” or “CNS” includes all cells and tissueof the brain and spinal cord of a vertebrate.

The term “peripheral nervous system” refers to all cells and tissue ofthe portion of the nervous system outside the brain and spinal cord,such as the motor neurons that mediate voluntary movement, the autonomicnervous system that includes the sympathetic nervous system and theparasympathetic nervous system and regulates involuntary functions, andthe enteric nervous system that controls the gastrointestinal system.Thus, the term “nervous system” includes, but is not limited to,neuronal cells, glial cells, astrocytes, cells in the cerebrospinalfluid (CSF), cells in the interstitial spaces, cells in the protectivecoverings of the spinal cord, epidural cells (i.e., cells outside of thedura mater), cells in non-neural tissues adjacent to or in contact withor innervated by neural tissue, cells in the epineurium, perineurium,endoneurium, funiculi, fasciculi, and the like.

As used herein, the term “patient” encompasses all mammalian species.

As used herein, the terms “treating” or “treatment” cover the treatmentof a disease-state in a mammal, particularly in a human, and include:(a) inhibiting the disease-state, i.e., arresting it development; and/or(b) relieving the disease-state, i.e., causing regression of the diseasestate.

As used herein, “prophylaxis” or “prevention” covers the preventivetreatment of a subclinical disease-state in a mammal, particularly in ahuman, aimed at reducing the probability of the occurrence of a clinicaldisease-state. Patients are selected for preventative therapy based onfactors that are known to increase risk of suffering a clinical diseasestate compared to the general population. “Prophylaxis” therapies can bedivided into (a) primary prevention and (b) secondary prevention.Primary prevention is defined as treatment in a subject that has not yetpresented with a clinical disease state, whereas secondary prevention isdefined as preventing a second occurrence of the same or similarclinical disease state.

As used herein, the term “risk reduction” covers therapies that lowerthe incidence of development of a clinical disease state. As such,primary and secondary prevention therapies are examples of riskreduction.

As used herein, the phrase “prophylactically effective amount” isintended to include an amount of an ASIC1a inhibitor and/or ananti-clotting agent described in the present application that iseffective to prevent or reduce the incidence of a TIA. When applied to acombination, the term refers to combined amounts of the activeingredients that result in a preventive effect, whether administered incombination, serially, or simultaneously.

As used herein, the phrase “therapeutically effective amount” isintended to include an amount of an ASIC1a inhibitor and/or ananti-clotting agent described in the present application that iseffective to treat a subject that has had a TIA and/or effective toprevent and/or reduce the incidence of stroke. When applied to acombination, the term refers to combined amounts of the activeingredients that result in the preventive or therapeutic effect, whetheradministered in combination, serially, or simultaneously.

As used herein, the term “thrombosis” refers to formation or presence ofa thrombus (pl. thrombi): clotting within a blood vessel that may causeischemia or infarction of tissues supplied by the vessel.

The term “embolism”, as used herein, refers to sudden blocking of anartery by a clot or foreign material that has been brought to its siteof lodgment by the blood current.

The term “thromboembolism”, as used herein, refers to obstruction of ablood vessel with thrombotic material carried by the blood stream fromthe site of origin to plug another vessel.

The term “thromboembolic disorders” entails both “thrombotic” and“embolic” disorders (defined above).

The term “thromboembolic disorders” as used herein includes arterialcardiovascular thromboembolic disorders, venous cardiovascular orcerebrovascular thromboembolic disorders, and thromboembolic disordersin the chambers of the heart or in the peripheral circulation. The term“thromboembolic disorders” as used herein also includes specificdisorders selected from, but not limited to, unstable angina or otheracute coronary syndromes, atrial fibrillation, first or recurrentmyocardial infarction, ischemic sudden death, transient ischemic attack,stroke, atherosclerosis, peripheral occlusive arterial disease, venousthrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism,coronary arterial thrombosis, cerebral arterial thrombosis, cerebralembolism, kidney embolism, pulmonary embolism, valvular heart disease,ventricular fibrillation, and thrombosis resulting from medicalimplants, devices, or procedures in which blood is exposed to anartificial surface that promotes thrombosis.

The present application provides methods and compositions forpreventing, treating and/or reducing the risk of transient ischemicattacks (TIAs) or ischemic stroke.

One aspect of the present application related to a method for preventingor reducing the incidence of a TIA in a subject at risk for developing aTIA, comprising orally administering to the subject a prophylacticallyeffective amount of a pharmaceutical composition comprising an ASIC1ainhibitor capable of penetrating the blood-brain barrier.

ASIC Inhibitors, Amiloride and Amiloride Analogs

The term “ASIC1a”, as used herein, refers to an ASIC1a protein orchannel from any species. The expression “ASIC1a inhibitor” refers to aproduct which inhibits acid sensing ion channel 1a (ASIC1a). Forexample, an exemplary human ASIC1a protein/channel is described inWaldmann, R., et al. 1997, Nature 386, pp. 173-177.

The ASIC1a inhibitor may be selective within the ASIC family. Selectiveinhibition of ASIC1a, as used herein, is inhibition that issubstantially stronger on ASIC1a than on another ASIC family member(s)when compared (for example, in cultured cells) after exposure of each tothe same (sub-maximal) concentration(s) of an inhibitor. The inhibitormay inhibit ASIC1a selectively relative to at least one other ASICfamily member (ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC 4, etc.) and/orselectively relative to every other ASIC family member. The strength ofinhibition for a selective inhibitor may be described by an inhibitorconcentration at which inhibition occurs (e.g., an ICso (inhibitorconcentration that produces 50% of maximal inhibition) or a K1 value(inhibition constant or dissociation constant)) relative to differentASIC family members. An ASIC1a-selective inhibitor may inhibit ASIC1aactivity at a concentration that is at least about two-, four-, orten-fold lower (one-half, one-fourth, or one-tenth the concentration orlower) than for inhibition of at least one other or of every other ASICfamily member. Accordingly, an ASIC₁a-selective inhibitor may have anIC₅₀ and/or K_(i) for ASIC1a inhibition that is at least about two-,four-, ten-, or twenty-fold lower (one-half, one-fourth, one-tenth,one-twentieth or less) than for inhibition of at least one other ASICfamily member and/or for inhibition of every other ASIC family member.

Accordingly, an ASIC 1a-specific inhibitor may have an IC₅₀ and/or K_(i)for ASIC1a relative to every other member of the ASIC family that is atleast about twenty-fold lower (five percent or less), such that, forexample, inhibition of other ASIC family members is at leastsubstantially (or completely) undetectable. In some embodiments, theASIC 1a-selective inhibitor has increased potency to homomeric ASIC1achannel and increased aqueous solubility comparing to any commerciallyavailable amiloride-related ASIC1a inhibitor.

In one embodiment, the ASIC1a inhibitor is selected from the groupconsisting of amiloride, amiloride analogs, and pharmaceuticallyacceptable salts or solvates thereof.

In one embodiment, the ASIC1a inhibitor is amiloride or apharmaceutically acceptable salt or solvate thereof. Amiloride, aguanidinium group containing pyrazine derivative, has been used for thetreatment of mild hypertension with little reported side effect.Amiloride works by directly blocking the epithelial sodium channel(ENaC) thereby inhibiting sodium reabsorption in the late distalconvoluted tubules, connecting tubules, and collecting ducts in thekidneys. This promotes the loss of sodium and water from the body, butwithout depleting potassium. As used herein, the term “amiloride” refersto both amiloride and salts of amiloride, such as amiloridehydrochloride.

In another embodiment, the ASIC1a inhibitor is an amiloride analog or apharmaceutically acceptable salt or solvate thereof. Amiloride analogs,as used herein, refer to chemical compounds having biological activitiessimilar to those of amiloride but with a slightly altered chemicalstructure.

In some embodiments, the amiloride analogs do not to block the humanNa⁺/Ca²⁺ ion exchanger. In other embodiments, the amiloride analog is aweak inhibitor of the Na⁺/Ca²⁺ ion exchanger and helps to maintain lowlevels of intracellular Ca′. In other embodiments, the amiloride analogis a very weak inhibitor of the Na⁺/Ca²⁺ ion exchanger with an IC₅₀ of1.1 mM or less. In other embodiments, the amiloride analogs do not blockthe ASIC2a and/or ASIC3 channels. In one embodiment, the amilorideanalog has increased selectivity for ASIC1a over the ASIC3 channeland/or ASIC2 channel.

Exemplary amiloride analogs for use in the present application include,but are not limited to benzamil, bepridil, KB-R7943, phenamil,5-(N—N-dimethyl) amiloride (DMA), 5-(N,N-hexamethylene) amiloride (HMA),5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N-methyl-N-isopropyl (MIA),pharmaceutically acceptable salts or solvates thereof, methylatedanalogs thereof, and combinations thereof.

In some embodiments, the amiloride analog has a hydrophobic substituentat the C₅—NH₂ position and/or on the guanidino group, as shown in FIG.21 below. In other embodiments, the amiloride analog is selected fromthe group consisting of methylated analogs of benzamil, amilorideanalogs containing a ring formed on a guanidine group, amiloride analogscontaining an acylguanidino group, and amiloride analogs containing awater solubilizing group formed on a guanidine group, wherein the watersolubilizing group is a N,N-dimethyl amino group or a sugar group.

Any suitable ASIC inhibitor or combination of inhibitors may be used.For example, a subject may be treated with an ASIC1a-selective inhibitorand a nonselective ASIC inhibitor, or with an ASIC1a-selective inhibitorand an inhibitor to a non-ASIC channel protein, such as a non-ASICcalcium channel. In some examples, a subject may be treated with anASIC1a-selective inhibitor and an inhibitor of NMDA receptors, such as aglutamate antagonist.

In other embodiments, the ASIC1a inhibitor is a peptide. The peptide mayhave any suitable number of amino acid residues, generally at leastabout ten, but less than a thousand residues, and more typically lessthan a hundred residues. In some embodiments, the peptide may have acystine knot motif. A cystine knot, as used herein, generally includesan arrangement of six or more cysteines. A peptide with these cysteinesmay create a “knot” including (1) a ring formed by two disulfide bondsand their connecting backbone segments, and (2) a third disulfide bondthat threads through the ring. In some examples, the peptide may be aconotoxin from an arachnid and/or cone snail species. For example, thepeptide may be PcTx1 (psalmotoxin 1), a toxin from a tarantula(Psalmopoeus cambridgei (Pc)). In other embodiments, the peptide may beone of four disulfide-rich spider venom peptides from the Australianfunnel-web spider Hadronyche infensa (i.e., Hi1a, Hi1b, Hi1c, Hi1d),which contains two tandem PcTx1-like sequences joined by a short linker.

In some examples, the peptide may be structurally related to PcTx1, suchthat the peptide and PcTx1 differ by at least one deletion, insertion,and/or substitution of one or more amino acids. For example, the peptidemay have at least about 25% or at least about 50% sequence identity,and/or at least about 25% or at least about 50% sequence similarity withPcTx1 (see below). Further aspects of peptides that may be suitable asinhibitors are described below in Example 3.

Methods of alignment of amino acid sequences for comparison andgeneration of identity and similarity scores are well known in the art.Exemplary alignment methods that may be suitable include (Best Fit) ofSmith and Waterman, a homology alignment algorithm (GAP) of Needlemanand Wunsch, a similarity method (Tfasta and Fasta) of Pearson andLipman, and/or the like. Computer algorithms of these and otherapproaches that may be suitable include, but are not limited to:CLUSTAL, GAP, BESTFIT, BLASTP, FASTA, and TFASTA.

As used herein, “sequence identity” or “identity” in the context of twopeptides relates to the percentage of residues in the correspondingpeptide sequences that are the same when aligned for maximumcorrespondence. In some examples, peptide residue positions that are notidentical may differ by conservative amino acid substitutions, whereamino acid residues are substituted for other amino acid residues withsimilar chemical properties (e.g., charge or hydrophobicity) andtherefore are expected to produce a smaller (or no) effect on thefunctional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards, to give a “similarity” of the sequences, whichcorrects for the conservative nature of the substitutions. For example,each conservative substitution may be scored as a partial rather than afull mismatch, thereby correcting the percentage sequence identity toprovide a similarity score. The scoring of conservative substitutions toobtain similarity scores is well known in the art and may be calculatedby any suitable approach, for example, according to the algorithm ofMeyers and Miller, Computer Applic. Biol Sci., 4: 11-17 (1988), e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

Anti-Clotting Agents

In another aspect, the pharmaceutical composition comprising one or moreASIC1a inhibitors further includes one or more anti-clotting agents.Anti-clotting agents for use in the present application includeantiplatelet agents, anticoagulant agents, anti-arrhythmic agents, or acombination thereof. Use of these anti-clotting agents may furtherincrease the prophylactic and/or therapeutic efficacy of the ASIC1ainhibitor(s) administered to a subject in need thereof, eithercombinatorially or synergistically.

In one embodiment, the pharmaceutical composition further includes oneor more antiplatelet agents. Exemplary antiplatelet agents include, butare not limited to COX inhibitors, adenosine diphosphate (ADP) receptorinhibitors, phosphodiesterase inhibitors, Glycoprotein IIb/IIIainhibitors, adenosine reuptake inhibitors, thromboxane inhibitors, andcombinations thereof. Some of the antiplatelet agents have multiplemodes of action as reflected in the lists below.

COX inhibitors include acetylsalicylic acid (e.g., Aspirin) andtriflusal (e.g., Disgren, Grendis, Aflen and Triflux), whichirreversibly inhibit the COX-1 enzyme, prostaglandin-endoperoxidesynthase-1 (i.e., COX-1 or PTGS1) and modify the enzymatic activity ofthe COX-2 enzyme (i.e., COX-2 or PTGS2), as well reversible COX-2inhibitors targeting COX-2/PTGS2, such as celecoxib (e.g., Celebrex).

Adenosine diphosphate (ADP) receptor inhibitors for use in the presentapplication include reversible or irreversible anatagonists of P2Y₁₂ ADPreceptors. Exemplary ADP receptor inhibitors include thienopyridines,such as the irreversible P2Y₁₂ inhibitors, prasugrel, clopidogrel (e.g.,Plavix), and reversible P2PY12 inhibitors, such as ticagrelor (e.g.,Brilinta).

Phosphodiesterase inhibitors for use in the present application include,but are not limited to dipyridamole (e.g., Persantine), cilostazol(e.g., Pletal), triflusal (e.g., Disgren, Grendis, Aflen and Triflux),and vorapaxar (e.g., Zontivity).

Glycoprotein IIb/IIIa inhibitors for use in the present applicationinclude, but are not limited to abciximab (e.g., ReoPro), eptifibatide(e.g., Integrilin), ifetroban, iloprost, isocarbacyclin methyl ester,itazigrel, lamifiban, lifarizine, molsidomine, nifedipine, orbofiban,oxagrelate, roxifiban, and tirofiban (Aggrastat).

Adenosine reuptake inhibitors for use in the present applicationinclude, but are not limited to acadesine, acetate, barbiturates,benzodiazepines, calcium channel inhibitors, carbamazepine,carisoprodol, cilostazol (Pletal), cyclobenzaprine, dilazep, estradiol,ethanol, flumazenil, hexobendine, hydroxyzine, indomethacin, inosine,KF24345, meprobamate, nitrobenzylthioguanosine, nitrobenzylthioinosine,papaverine, pentoxifylline, phenothiazines, phenytoin, progesterone,propentofylline, propofol, puromycin, R75231, RE 102 BS, soluflazine,toyocamycin, tracazolate, and tricyclic antidepressants.

Thromboxane inhibitors for use in the present application inhibit thesynthesis of thromboxane and/or inhibit the target effect ofthromboxane. Exemplary thromboxane inhibitors include, but are notlimited to acetylsalicylic acid (e.g., Aspirin), dipyridamole,ifetroban, naproxen, picotamide, ridogrel, sulotroban, terutroban,ticlopidine, trapidil, triclopidine, trifenagrel, trifusal (e.g.,Disgren, Grendis, Aflen and Triflux), and trilinolein.

In another embodiment, the pharmaceutical composition further includesone or more anticoagulant agents. In a particular embodiment, theanticoagulant agent is a vitamin-K epoxide reductase inhibitor. In otherembodiments, the anticoagulant agent is a direct Factor Xa inhibitor, anindirect Factor Xa inhibitor, a direct thrombin inhibitor, or anindirect thrombin inhibitor (which are collectively known as direct oralanticoagulants (DOACs) or non-VKA oral anticoagulants). VitaminK-epoxide reductase inhibitors for use in combination with the ASIC1ainhibitors of the present application include 4-hydroxycoumarinderivatives and 1,3-indandione derivatives. Exemplary vitamin K epoxidereductase inhibitors include, but are not limited to acenocoumarol(e.g., Sintrom and Sinthrome), anisindione, clorindione, coumarin,coumadin (e.g., warfarin), dicumarol and its derivatives (e.g.,bis-hydroxycoumarin, bishydroxycoumarin, dicoumarin, dicoumarol),disulfiram, ethyl biscoumacetate, N-ethylmaleimide, fluindione,phenindione (e.g., Dindevan), phenprocoumon (e.g., Marcoumar, Marcumarand Falithrom), 1-N-methyl-5-thiotetrazole,5,5′-dithiobis(1-methyltetrazole), pharmaceutically acceptable salts andsolvates thereof, and combinations thereof.

Non-VKA oral anticoagulants (NOACs) for use in the present applicationinclude direct factor Xa inhibitors, such as apixaban (e.g., Eliquis),edoxaban (e.g., Savaysa, Lixiana), rivaroxaban (e.g., Xarelto),betrixaban (e.g., Bevyxxa), and YM466; direct thrombin inhibitors (orfactor IIa inhibitors), such as AZD-0837, dabigatran (e.g., Pradaxa,Pradax, and Prazaxa), ximelagatran (e.g., Exanta), and melagatran, theactive form of ximelagatran; indirect factor Xa inhibitors, such asfondaparinux (e.g., Arixtra), ultralow molecular weight heparin (ULMWH);indirect thrombin inhibitors, such as heparin, antithrombin, heparin incombination with antithrombin, enoxaparin (e.g., Lovenox), low molecularweight heparin (LMWH), dalteparin sodium (e.g., Fragmin), batroxobin,and hementin; including salts and solvates thereof, and combinationsthereof.

In another embodiment, the pharmaceutical composition for use in thepresent application further includes one or more anti-arrhythmic agents.Exemplary anti-arrhythmic agents include amiodarone, AZD-1305,budiodarone, celivarone, dofetilide, dronedarone, ibutilide, flecainide,propafenone, quinidine, sotolol, vernakalant, and combinations thereof.

Any single, plurality, or combination of anticoagulants, salts thereof,solvates thereof, and derivatives thereof, may be used for modulatinganticoagulant function, including other anticoagulants not mentionedhere without departing from the present application.

Administration of Inhibitors

Administration of the ASIC1a inhibitors and/or the additionalanti-clotting agents may be performed once or a plurality of times, andat any suitable time relative to TIA diagnosis, to alleviate the risk ofan additional TIA and/or stroke. Accordingly, administration may beperformed before (e.g., prophylactically) or after a TIA has beendetected, after a minor ischemic episode, during chronic ischemia, aftera full stroke, and/or the like.

In preferred embodiments, the ASIC1a inhibitors and/or anti-clottingagents of the present application are orally administered in aprophylactically effective amount (or simply “an effective amount”). Aprophylactically effective amount or an effective amount of an inhibitoror agent, as used herein, is any amount of the inhibitor or agent that,when administered to subjects, reduces, in a significant number of thesubjects, the degree, incidence, and/or extent of transient ischemicattacks (TIAs) in the subject. Accordingly, a prophylactically effectiveamount may be determined, for example, in clinical studies in whichvarious amounts of the inhibitor are administered to test subjects (and,generally, compared to a control group of subjects). Alternatively, oneor more of the anti-clotting agents may be administered in atherapeutically effective amounts and/or may be administeredintravenously, intramuscularly, intrathecally orintracerebroventricularly.

In some embodiments, the ASIC1a inhibitor and/or anti-clotting agentsare formulated, individually or in combination, for daily administrationin one or more doses in the range of 0.01-30 mg/kg body weight, 0.01-10mg/kg body weight, 0.01-3 mg/kg body weight, 0.01-1 mg/kg body weight,0.01-0.3 mg/kg body weight, 0.01-0.1 mg/kg body weight, 0.01-0.03 mg/kgbody weight, 0.03-30 mg/kg body weight, 0.03-10 mg/kg body weight,0.03-3 mg/kg body weight, 0.03-1 mg/kg body weight, 0.03-0.3 mg/kg bodyweight, 0.03-0.1 mg/kg body weight, 0.1-30 mg/kg body weight, 0.1-10mg/kg body weight, 0.1-3 mg/kg body weight, 0.1-1 mg/kg body weight,0.1-0.3 mg/kg body weight, 0.3-30 mg/kg body weight, 0.3-10 mg/kg bodyweight, 0.3-3 mg/kg body weight, 0.3-1 mg/kg body weight, 1-30 mg/kgbody weight, 1-10 mg/kg body weight, 1-3 mg/kg body weight, 3-30 mg/kgbody weight, 3-10 mg/kg body weight or 10-30 mg/kg body weight.

In other embodiments, the ASIC1a inhibitor and/or other anti-clottingagent(s) are formulated, individually or in combination, in one or moredoses in the range of 0.1-1000 mg/dose, 0.1-300 mg/dose, 0.1-100mg/dose, 0.1-30 mg/dose, 0.1-10 mg/dose, 0.1-3 mg/dose, 0.1-1 mg/dose,0.1-0.3 mg/dose, 0.3-1000 mg/dose, 0.3-300 mg/dose, 0.3-100 mg/dose,0.3-30 mg/dose, 0.3-10 mg/dose, 0.3-3 mg/dose, 0.3-1 mg/dose, 1-1000mg/dose, 1-300 mg/dose, 1-100 mg/dose, 1-30 mg/dose, 1-10 mg/dose, 1-3mg/dose, 3-1000 mg/dose, 3-300 mg/dose, 3-100 mg/dose, 3-30 mg/dose,3-10 mg/dose, 10-1000 mg/dose, 10-300 mg/dose, 10-100 mg/dose, 10-30mg/dose, 30-1000 mg/dose, 30-300 mg/dose, 30-100 mg/dose, 100-1000mg/dose, 100-300 mg/dose, or 300-1000 mg/dose.

The inhibitor(s) may be administered in any suitable form and in anysuitable composition to subjects. In some examples, the inhibitors maybe configured as a pharmaceutically acceptable salt or solvate. Thecomposition(s) may be formulated to include, for example, a fluidcarrier/solvent (a vehicle), a preservative, one or more excipients, acoloring agent, a flavoring agent, a salt(s), an anti-foaming agent,and/or the like. The inhibitor(s) may be present at a concentration inthe vehicle that provides a prophylactically or therapeuticallyeffective amount of the inhibitor(s) for prevention or treatment of TIAswhen administered to a subject at risk for developing a TIA or stroke.

In some embodiments, amiloride analogs with higher water solubility orlipid solubility are used. For example, the amiloride analog may containa water solubilizing group, such as an NN-dimethyl amino group or asugar, at the guanidino group to improve water solubility (formula13-16, FIG. 24). In some embodiments, the amiloride analog has a watersolubility of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80mM, 90 mM, 100 mM or higher. In other embodiments, the amiloride analoghas a solubility that allows for a 10 mg, 25 mg, 50 mg, 100 mg, 150 mg,200 mg, 250 mg, 300 mg, 400 mg, or 500 mg dose for oral administration.

Generally, the pharmaceutical compositions of the present applicationinclude one or more pharmaceutically acceptable carrier(s). As usedherein, a “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, sweeteners and thelike. The pharmaceutically acceptable carriers may be prepared from awide range of materials including, but not limited to flavoring agents,sweetening agents and miscellaneous materials such as buffers andabsorbents that may be needed in order to prepare a particulartherapeutic composition. The use of such media and agents withpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the prophylactic or therapeuticcompositions is contemplated. Optionally, the amiloride and/or anamiloride analog may be combined with other active ingredients, whichare not contraindicated by the ASIC1a inhibitor(s), including amilorideand/or its analog(s), and which further increase the prophylactic and/ortherapeutic efficacy of the ASIC1a inhibitor(s).

In preferred embodiments, the pharmaceutical composition is formulatedfor oral administration. In particular embodiments, the pharmaceuticalcomposition is provided in a dry form, and is formulated into a tabletor capsule form. Tablets may be formulated in accordance withconventional procedures employing solid carriers well-known in the art.Hard and soft capsules employed in the present invention can be madefrom any pharmaceutically acceptable material, such as gelatin orcellulosic derivatives.

In other embodiments, the pharmaceutical composition is formulated forintravenous injection, intramuscular injection, intrathecal injection orintracerebroventricular injection. Pharmaceutical compositions suitablefor injectable use include sterile aqueous solutions (where watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the injectablecompositions are sterile and are fluid to the extent that easysyringability exists. The injectable composition is preferably stableunder the conditions of manufacture and storage and preserved againstthe contaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requitedparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activeagent(s) in the required amount(s) in an appropriate solvent, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the active,ingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In certain embodiments, the pharmaceutical composition is formulated forcontrolled release of the ASIC1a inhibitor and/or anti-clotting agentsin the composition. A controlled release formulation may be designed forimmediate release, extended-release, delayed-release or a combinationthereof. Extended-release, also known as sustained-release, time-releaseor timed-release, controlled-release (CR), modified release (MR), orcontinuous-release (CR or Contin), provides a mechanism for the slowrelease over time of one or more active agents, typically in an oralformulation comprising tablets or capsules to dissolve slowly andrelease the active ingredient over time. The advantages ofsustained-release tablets or capsules are that they can often be takenless frequently than instant-release formulations of the same drug, andthat they keep steadier levels of the drug in the bloodstream, thusextending the duration of the drug action.

In one embodiment, the pharmaceutical composition is formulated forextended release by embedding the active ingredient in a matrix ofinsoluble substance(s) such as acrylics or chitin. An extended releaseform is designed to release the active ingredient at a predeterminedrate by maintaining a constant drug level for a specific period of time.

In another embodiment, the pharmaceutical composition is formulated fordelayed-release, such that the active ingredient(s) is not immediatelyreleased upon administration. A non-limiting example of a delayedrelease vehicle is an enteric coated oral medication that dissolves inthe intestines rather than the stomach.

In other embodiments, the pharmaceutical composition is formulated forimmediate release of a portion of the active ingredient(s), followed byextended-release of the remainder of the active ingredient(s). In oneembodiment, the pharmaceutical composition is formulated as a powderthat can be ingested for rapid release of the active ingredient. Inanother embodiment, the pharmaceutical composition is formulated into aliquid, gel, liquid suspension or emulsion form. Said liquid, gel,suspension or emulsion may be ingested by the subject in naked form orcontained within a capsule.

In yet another embodiment, the pharmaceutical composition may beprovided as a skin or transdermal patch for the topical administrationof controlled and/or sustained quantities of the active ingredient.

Methods for Preventing or Treating Disease

One aspect of the present application relates to a method for preventingor treating a transient ischemic attack (TIA) in a subject. In oneembodiment, the method includes orally administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising an ASIC1a inhibitor as described above. In other embodiments,the pharmaceutical composition is administered intravenously,intramuscularly, intrathecally or intracerebroventricularly.

The method and pharmaceutical composition of the present application canbe used in any subject at risk for developing TIAs and/or stroke.

Risk factors include nonmodifiable factors (age, sex, race, andsignificant family history); modifiable factors (body weight,hypertension, unhealthy lipid profile, cerebral microbleeds,cardiovascular diseases, including coronary artery disease, myocardialinfarction, peripheral arterial disease, valvular disease, atrialfibrillation, atrial flutter; diabetes mellitus; and lifestyle choices(cigarette smoking, alcohol consumption, illicit drug use, unhealthydiet/poor nutrition, and physical inactivity).

In one embodiment, the subject is at risk for developing a TIA orstroke.

In another embodiment, the subject has recently had heart surgery or hasbeen previously diagnosed as having had a TIA or stroke.

In another embodiment, subject has an abnormal heart rhythm selectedfrom the group consisting of atrial fibrillation, atrial flutter,ventricular tachycardia, and ventricular fibrillation.

In another embodiment, the compositions of the present application maybe used in a method for treatment of a thromboembolic disorder selectedfrom the group consisting of acute coronary syndrome, arterial embolism,atherosclerosis, atrial fibrillation, carotid artery disease, cerebralarterial thrombosis, cerebral embolism, coronary arterial thrombosis,coronary heart disease, deep vein thrombosis, kidney embolism,myocardial infarction, peripheral arteriopathy, pulmonary embolism,stroke, thrombophlebitis, thrombosis, transient ischemic attack,unstable angina, valvular heart disease, ventricular fibrillation, andvenous thrombosis or combination thereof.

In another embodiment, the subject has diabetes or sickle cell disease.

The subject may be an animal subject or a human subject. The term“animal”, as used herein, refers to any animal that is not human.Exemplary animals that may be suitable include any animal with abloodstream, such as rodents (mice, rats, etc.), dogs, cats, birds,sheep, goats, non-human primates, etc. The animal may be treated for itsown sake, e.g., for veterinary purposes (such as treatment of a pet).Alternatively, the animal may provide an animal model of nerve injury,such as ischemia, to facilitate testing drug candidates for human use,such as to determine the candidates' potency, window of effectiveness,side effects, etc.

In another aspect, the present application provides a method forpreventing or treating a nerve injury in a subject. In one embodiment,the method includes orally administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising an ASIC1a inhibitor as described above. In other embodiments,the pharmaceutical composition is administered intravenously,intramuscularly, intrathecally or intracerebroventricularly.

In some embodiments, the subject has ischemia, an ischemia-relatedcondition, a history of ischemia, and/or a significant chance ofdeveloping ischemia after prophylactic or therapeutic treatment hasbegun and during a time period in which prevention or treatment is stilleffective.

Subjects for prevention and/or treatment may be selected by any suitablecriteria. Exemplary criteria may include any detectable symptoms ofischemia, a history of ischemia, an event that increases the risk of (orinduces) ischemia (such as a surgical procedure, trauma, administrationof a medication, etc.), and/or the like. A history of ischemia mayinvolve one or more prior ischemic episodes. In some examples, a subjectselected for treatment may have had an onset of ischemia that occurredat least about one, two, or three hours before treatment begins, or aplurality of ischemic episodes (such as transient ischemic attacks) thatoccurred less than about one day, twelve hours, or six hours prior toinitiation of treatment.

As used herein, the term “nerve injury” means an acute or chronic injuryto or adverse condition of a nervous system tissue or cell resultingfrom physical transaction or trauma, contusion or compression orsurgical lesion, vascular pharmacologic insults including hemorrhagic orischemic damage, or from neurodegenerative or any other neurologicaldisease, or any other factor causing the injury to or adverse conditionof the nervous system tissue or cell. In some embodiments, the nerveinjury is caused by cognitive disorders, psychotic disorders,neurotransmitter-mediated disorders or neuronal disorders. Nerve injuryincludes injuries to the nervous system (i.e., nervous system injuries)and brain injury.

As used herein, the term “cognitive disorders” refers to and intendsdiseases and conditions that are believed to involve or be associatedwith or do involve or are associated with progressive loss of structureand/or function of neurons, including death of neurons, and where acentral feature of the disorder may be the impairment of cognition(e.g., memory, attention, perception and/or thinking). These disordersinclude pathogen-induced cognitive dysfunction, e.g., HIV associatedcognitive dysfunction or Lyme disease associated cognitive dysfunction.In some embodiments, the cognitive disorders are degenerative cognitivedisorders. Examples of degenerative cognitive disorders includeAlzheimer's Disease, Huntington's Disease, Parkinson's Disease,amyotrophic lateral sclerosis (ALS), autism, mild cognitive impairment(MCI), stroke, traumatic brain injury (TBI), age-associated memoryimpairment (AAMI) and epilepsy.

As used herein, the term “psychotic disorders” refers to and intendsmental diseases or conditions that are believed to cause or do causeabnormal thinking and perceptions. Psychotic disorders are characterizedby a loss of reality which may be accompanied by delusions,hallucinations (perceptions in a conscious and awake state in theabsence of external stimuli which have qualities of real perception, inthat they are vivid, substantial, and located in external objectivespace), personality changes and/or disorganized thinking. Other commonsymptoms include unusual or bizarre behavior, as well as difficulty withsocial interaction and impairment in carrying out the activities ofdaily living. Exemplary psychotic disorders are schizophrenia, bipolardisorders, psychosis, anxiety, depression and chronic pain.

As used herein, the term “neurotransmitter-mediated disorders” refers toand intends diseases or conditions that are believed to involve or beassociated with or do involve or are associated with abnormal levels ofneurotransmitters such as histamine, glutamate, serotonin, dopamine,norepinephrine or impaired function of aminergic G protein-coupledreceptors. Exemplary neurotransmitter-mediated disorders include spinalcord injury, diabetic neuropathy, allergic diseases and diseasesinvolving geroprotective activity such as age-associated hair loss(alopecia), age-associated weight loss and age-associated visiondisturbances (cataracts). Abnormal neurotransmitter levels areassociated with a wide variety of diseases and conditions including, butnot limited, to Alzheimer's disease, Parkinson's Disease, autism,Guillain-Barre syndrome, mild cognitive impairment, schizophrenia,anxiety, multiple sclerosis, stroke, traumatic brain injury, spinal cordinjury, diabetic neuropathy, fibromyalgia, bipolar disorders, psychosis,depression and a variety of allergic diseases.

As used herein, the term “neuronal disorders” refers to and intendsdiseases or conditions that are believed to involve, or be associatedwith, or do involve or are associated with neuronal cell death and/orimpaired neuronal function or decreased neuronal function. Exemplaryneuronal indications include neurodegenerative diseases and disorderssuch as Alzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis (ALS), Parkinson's disease, canine cognitive dysfunctionsyndrome (CCDS), Lewy body disease, Menkes disease, Wilson disease,Creutzfeldt-Jakob disease, Fahr disease, an acute or chronic disorderinvolving cerebral circulation, such as ischemic or hemorrhagic strokeor other cerebral hemorrhagic insult, age-associated memory impairment(AAMI), mild cognitive impairment (MCI), injury-related mild cognitiveimpairment (MCI), post-concussion syndrome, post-traumatic stressdisorder, adjuvant chemotherapy, traumatic brain injury (TBI), neuronaldeath mediated ocular disorder, macular degeneration, age-relatedmacular degeneration, autism, including autism spectrum disorder,Asperger syndrome, and Rett syndrome, an avulsion injury, a spinal cordinjury, myasthenia gravis, Guillain-Barre syndrome, multiple sclerosis,diabetic neuropathy, fibromyalgia, neuropathy associated with spinalcord injury, schizophrenia, bipolar disorder, psychosis, anxiety ordepression, and chronic pain.

In some embodiments, the nerve injuries or nervous system injuries arecaused by a change in the ion flux into neurons or a nervous systemtissue. As used herein, the term “nervous system tissue” refers toanimal tissue comprising nerve cells, neuropils, glia, neuralinflammatory cells, and endothelial cells in contact with “nervoussystem tissue”. “Nerve cells” may be any type of nerve cell known tothose of skill in the art including, but not limited to neurons. As usedherein, the term “neuron” represents a cell of ectodermal embryonicorigin derived from any part of the nervous system of an animal. Neuronsexpress well-characterized neuron-specific markers, includingneurofilament proteins, NeuN (Neuronal Nuclei marker), MAP2, and classIII tubulin. Included as neurons are, for example, hippocampal,cortical, midbrain dopaminergic, spinal motor, sensory, enteric,sympathetic, parasympathetic, septal cholinergic, central nervous systemand cerebellar neurons. “Glial cells” useful in the present inventioninclude, but are not limited to astrocytes, Schwan cells, andoligodendrocytes. “Neural inflammatory cells” useful in the presentapplication include, but are not limited to cells of myeloid originincluding macrophages and microglia.

In some embodiments, the pharmaceutical compositions and methods of thepresent application relate to reducing nerve injuries caused by ischemiaor an ischemia-related condition. Ischemia, as used herein, is a reducedblood flow to an organ(s) and/or tissue(s). The reduced blood flow maybe caused by many mechanisms, including but are not limited to, apartial or complete blockage (an obstruction), a narrowing (aconstriction), and/or a leak/rupture, of one or more blood vessels thatsupply blood to the organ(s) and/or tissue(s). Ischemia may be createdby thrombosis, an embolism, atherosclerosis, hypertension, hemorrhage,an aneurysm, surgery, trauma, medication, and the like. The reducedblood flow thus may be chronic, transient, acute or sporadic.

An ischemia-related condition may be any consequence of ischemia. Theconsequence may be substantially concurrent with the onset ischemia(e.g., a direct effect of the ischemia) and/or may occur substantiallyafter ischemia onset and/or even after the ischemia is over (e.g., anindirect, downstream effect of the ischemia, such reperfusion of tissuewhen ischemia ends). Exemplary ischemia-related conditions may includeany combination of the symptoms (and/or conditions) listed above.Alternatively, or in addition, the symptoms may include local and/orsystemic acidosis (pH decrease), hypoxia (oxygen decrease), free radicalgeneration, and/or the like.

In some embodiments, the ischemia-related condition is stroke. Stroke,as used herein, is brain ischemia produced by a reduced blood supply toa part (or all) of the brain. Symptoms produced by stroke may be sudden(such as loss of consciousness) or may have a gradual onset over hoursor days. Furthermore, the stroke may be a major ischemic attack (a fullstroke) or a more minor, transient ischemic attack, among others.Symptoms produced by stroke may include, for example, hemiparesis,hemiplegia, one-sided numbness, one-sided weakness, one-sided paralysis,temporary limb weakness, limb tingling, confusion, trouble speaking,trouble understanding speech, trouble seeing in one or both eyes, dimvision, loss of vision, trouble walking, dizziness, a tendency to fall,loss of coordination, sudden severe headache, noisy breathing, and/orloss of consciousness. Alternatively, or in addition, the symptoms maybe detectable more readily or only via tests and/or instruments, forexample, an ischemia blood test (e.g., to test for altered albumin,particular protein isoforms, damaged proteins, etc.), anelectrocardiogram, an electroencephalogram, an exercise stress test,brain CT or Mill scanning and/or the like.

Acid-base balance is important for biological systems. Normal brainfunction depends on the complete oxidation of glucose, with the endproduct of CO₂ and H₂O for its energy requirements. During ischemia,increased anaerobic glycolysis, due to the lack of oxygen supply, leadsto lactic acid accumulation. Accumulation of lactic acid, along withincreased H⁺ release from ATP hydrolysis, causes decreases in tissue pH.Extracellular pH (pH_(o)) typically falls to 6.5 during ischemia, and itcan fall below 6.0 during severe ischemia or under hyperglycemicconditions.

Any organ or tissue may experience a reduced blood flow and requiredtreatment for ischemia. Exemplary organs and/or tissues include, but arenot limited to, brain, arteries, heart, intestines and eye (e.g., theoptic nerve). Ischemia-induced injuries (i.e., disease and/or damageproduced by various types of ischemia) include, but are not limited to,ischemic myelopathy, ischemic optic neuropathy, ischemic colitis,coronary heart disease, and/or cardiac heart disease (e.g., angina,heart attack, etc.), among others. Ischemia-induced injury thus maydamage and/or kill cells and/or tissue, for example, producing necrotic(infarcted) tissue, inflammation, and/or tissue remodeling, amongothers, at affected sites within the body. Treatment according toaspects of the present application may reduce the incidence, extent,and/or severity of this injury.

In some embodiments, the amiloride, amiloride analog or apharmaceutically acceptable salt or solvate thereof is given in a doserange of 0.1 mg-10 mg/kg body weight. In other embodiments, thepharmaceutical composition is administered within one hour of the onsetof an ischemic event, within five hours of the onset of an ischemicevent, or between one hour and five hours of the onset of an ischemicevent.

FIG. 1 shows a flowchart 20 with exemplary steps 22, 24 that may beperformed in a method of reducing nerve injury in an ischemic subject.The steps may be performed any suitable number of times and in anysuitable combination. In the method, an ischemic subject (or subjects)may be selected for treatment, indicated at 22. An ASIC-selectiveinhibitor then may be administered to the ischemic subject(s), indicatedat 24. Administration of the inhibitor to the ischemic subject may be ina therapeutically effect amount, to reduce ischemia-induced injury tothe subject, for example, reducing the amount of brain damage resultingfrom a stroke.

A potential explanation for the efficacy of the ischemia treatment ofFIG. 1 may be offered by the data of the present teachings (e.g., seeExample 1). In particular, the damaging effects of ischemia may not beequal to acidosis, that is, acidification of tissue/cells via ischemiamay not be sufficient to produce ischemia-induced injury. Instead,ischemia-induced injury may be caused, in many cases, by calcium fluxinto cells mediated by a member(s) of the ASIC family, particularlyASIC1a. Accordingly, selective inhibition of the channel activity ofASIC1a may reduce this harmful calcium flux, thereby reducingischemia-induced injury.

FIG. 2 shows a flowchart 30 with exemplary steps 32, 34 that may beperformed in a method of identifying drugs for treatment of ischemia.The steps may be performed any suitable number of times and in anysuitable combination. In the method, one or more ASIC-selectiveinhibitors may be obtained, indicated at 32. The inhibitors then may betested on an ischemic subject for an effect on ischemia-induced injury,indicated at 34.

EXAMPLES

The following examples describe selected aspects and embodiments of thepresent teachings, particularly data describing in vitro and in vivoeffects of ASIC inhibition. These examples are intended for the purposesof illustration and should not be construed to limit the scope of thepresent teachings.

Example 1: Neuroprotection in Ischemia by Blocking Calcium-PermeableAcid-Sensing Ion Channels

This example describes experiments showing a role of ASIC1a in mediatingischemic injury and the ability ASIC1a inhibitors to reduce ischemicinjury; see FIGS. 2-10. Ca²⁺ toxicity may play a central role inischemic brain injury. The mechanism by which toxic Ca²⁺ loading ofcells occurs in the ischemic brain has become less clear as multiplehuman trials of glutamate antagonists have failed to show effectiveneuroprotection in stroke. Acidosis is a common feature of ischemia andplays a critical role in brain injury. This example demonstrates thatacidosis activates Ca²⁺-permeable acid-sensing ion channels (ASICs),which may induce glutamate receptor-independent, Ca²⁺-dependent,neuronal injury. Accordingly, cells lacking endogenous ASICs may beresistant to acid injury, while transfection of Ca²⁺-permeable ASIC1amay establish sensitivity. In focal ischemia, intracerebroventricularinjection of ASIC1a inhibitors or knockout of the ASIC1a gene mayprotect the brain from ischemic injury and may do so more potently thanglutamate antagonism.

The normal brain requires complete oxidation of glucose to fulfill itsenergy requirements. During ischemia, oxygen depletion forces the brainto switch to anaerobic glycolysis. Accumulation of lactic acid as abyproduct of glycolysis and protons produced by ATP hydrolysis causes pHto fall in the ischemic brain and aggravates ischemic brain injury.

Acid-sensing ion channels (ASICs) are a class of ligand-gated channelsexpressed throughout neurons of mammalian central and peripheral nervoussystems. To date, six ASIC subunits have been cloned. Four of thesesubunits form functional homomultimeric channels that are activated byacidic pH to conduct a sodium-selective, amiloride-sensitive, cationcurrent. Two of the ASIC subunits, ASIC1a and ASIC2a subunits, have beenshown to be abundant in the brain.

Experimental Procedures Neuronal Culture

Following anesthesia with halothane, cerebral cortices were dissectedfrom E16 Swiss mice or P1 ASIC1^(+/+) and ASIC1^(−/−) mice and incubatedwith 0.05% trypsin-EDTA for 10 min at 37° C. Tissues were thentriturated with fire-polished glass pipettes and plated onpoly-L-ornithine-coated 24-well plates or 25×25 mm glass coverslips at adensity of 2.5×10⁵ cells per well or 10⁶ cells per coverslip. Neuronswere cultured with MEM supplemented with 10% horse serum (for E16cultures) or Neurobasal medium supplemented with B27 (for P1 cultures)and used for electrophysiology and toxicity studies after 12 days. Glialgrowth was suppressed by addition of 5-fluoro-2-deoxyuridine anduridine, yielding cultured cells with 90% neurons as determined by NeuNand GFAP staining (data not shown).

Electrophysiology

ASIC currents were recorded with whole-cell patch-clamp andfast-perfusion techniques. The normal extracellular solution (ECF)contained (in mM) 140 NaCl, 5.4 KCl, 25 HEPES, 20 glucose, 1.3 CaCl₂),1.0 MgCl2, 0.0005 TTX (pH 7.4), 320-335 mOsm. For low pH solutions,various amounts of HCl were added. For solutions with pH<6.0, IVIESinstead of HEPES was used for more reliable pH buffering. Patchelectrodes contained (in mM) 140 CsF, 2.0 MgCl₂, 1.0 CaC², 10 HEPES, 11EGTA, 4 MgATP (pH 7.3), 300 mOsm. The Na⁺-free solution consisted of 10mM CaCl₂), 25 mM HEPES with equiosmotic NMDG or sucrose substituting forNaCl (Chu et al., 2002). A multibarrel perfusion system (SF-77B, WarnerInstrument Co.) was employed for rapid exchange of solutions.

Cell Injury Assay—LDH Measurement

Cells were washed three times with ECF and randomly divided intotreatment groups. MK801 (10 μM), CNQX (20 μM), and nimodipine (5 μM)were added in all groups to eliminate potential secondary activation ofglutamate receptors and voltage-gated Ca²⁺ channels. Following acidincubation, neurons were washed and incubated in Neurobasal medium at37° C. LDH release was measured in culture medium using the LDH assaykit (Roche Molecular Biochemicals). Medium (100 μL) was transferred fromculture wells to 96-well plates and mixed with 100 μL reaction solutionprovided by the kit. Optical density was measured at 492 nm 30 minlater, utilizing a microplate reader (Spectra Max Plus, MolecularDevices). Background absorbance at 620 was subtracted. The maximalreleasable LDH was obtained in each well by 15 min incubation with 1%Triton X-100 at the end of each experiment.

Ca²⁺ Imaging

Cortical neurons grown on 25×25 mm glass coverslips were washed threetimes with ECF, incubated with 5 μM fura-2-acetoxymethyl ester for 40min at 22° C., washed three times, and then incubated in normal ECF for30 min. Coverslips were transferred to a perfusion chamber on aninverted microscope (Nikon TE300). Cells were illuminated using a xenonlamp and observed with a 40× UV fluor oil-immersion objective lens, andvideo images were obtained using a cooled CCD camera (Sensys KAF 1401,Photometrics). Digitized images were acquired and analyzed in a PCcontrolled by Axon Imaging Workbench software (Axon Instruments). Theshutter and filter wheel (Lambda 10-2) were controlled by the softwareto allow timed illumination of cells at 340 or 380 nm excitationwavelengths. Fura-2 fluorescence was detected at emission wavelength of510 nm. Ratio images (340/380) were analyzed by averaging pixel ratiovalues in circumscribed regions of cells in the field of view. Thevalues were exported to SigmaPlot for further analysis.

Fluorescein-Diacetate Staining and Propidium Iodide Uptake

Cells were incubated in ECF containing fluorescein-diacetate (FDA) (5μM) and propidium iodide (PI) (2 μM) for 30 min followed by wash withdye-free ECF. Alive (FDA-positive) and dead (PI-positive) cells wereviewed and counted on a microscope (Zeiss) equipped with epifluorescenceat 580/630 nm excitation/emission for PI and 500/550 nm for FDA. Imageswere collected using an Optronics DEI-730 camera equipped with a BQ 8000sVGA frame grabber and analyzed using computer software (Bioquant, TN).

Transfection of COS-7 Cells

COS-7 cells were cultured in MEM with 10% HS and 1% PenStrep (GIBCO). At−50% confluence, cells were cotransfected with cDNAs for ASICs and GFPin pc^(DNA3) vector using FuGENE6 transfection reagents (Roche MolecularBiochemicals). DNA for ASICs (0.75 μg) and 0.25 μg of DNA for GFP wereused for each 35 mm dish. GFP-positive cells were selected forpatch-clamp recording 48 hr after transfection. For stable transfectionof ASIC1a, 500 m/mL G418 was added to culture medium I week followingthe transfection. The surviving G418-resistant cells were further platedand passed for >5 passages in the presence of G418. Cells were thenchecked with patch-clamp and immunofluorescent staining for theexpression of ASIC1a.

Oxygen-Glucose Deprivation

Neurons were washed three times and incubated with glucose-free ECF atpH 7.4 or 6.0 in an anaerobic chamber (Model 1025, Forma Scientific)with an atmosphere of 85% N₂, 10% H₂, and 5% CO₂ at 35° C.Oxygen-glucose deprivation (OGD) was terminated after 1 hr by replacingthe glucose-free ECF with Neurobasal medium and incubating the culturesin a normal cell culture incubator. With HEPES-buffered ECF used, 1 hrOGD slightly reduced pH from 7.38 to 7.28 (n=3) and from 6.0 to 5.96(n=4).

Focal Ischemia

Transient focal ischemia was induced by suture occlusion of the middlecerebral artery (MCAO) in male rats (SD, 250-300 g) and mice (withcongenic C57B16 background, ˜25 g) anesthetized using 1.5% isoflurane,70% N₂O, and 28.5% O₂ with intubation and ventilation. Rectal andtemporalis muscle temperature was maintained at 37° C.±0.5° C. with athermostatically controlled heating pad and lamp. Cerebral blood flowwas monitored by transcranical LASER doppler. Animals with blood flownot reduced below 20% were excluded.

Animals were killed with chloral hydrate 24 hr after ischemia. Brainswere rapidly removed, sectioned coronally at 1 mm (mice) or 2 mm (rats)intervals, and stained by immersion in vital dye (2%)2,3,5-triphenyltetrazolium hydrochloride (TTC). Infarction area wascalculated by subtracting the normal area stained with TTC in theischemic hemisphere from the area of the nonischemic hemisphere. Infarctvolume was calculated by summing infarction areas of all sections andmultiplying by slice thickness. Rat intraventricular injection wasperformed by stereotaxic technique using a microsyringe pump withcannula inserted stereotactically at 0.8 mm posterior to bregma, 1.5 mmlateral to midline, and 3.8 mm ventral to the dura. All manipulationsand analyses were performed by individuals blinded to treatment groups.

Results

(a) Acidosis Activates ASICs in Mouse Cortical Neurons

FIGS. 3 and 4 shows exemplary data related to the electrophysiology andpharmacology of ASICs in cultured mouse cortical neurons. FIGS. 3A and3B are graphs illustrating the pH dependence of ASIC currents activatedby a pH drop from 7.4 to the pH values indicated. Dose-response curveswere fit to the Hill equation with an average pH_(0.5) of 6.18±0.06(n=10). FIGS. 3C and 3D are graphs illustrating the current-voltagerelationship of ASICs (n=5). The amplitudes of ASIC current at variousvoltages were normalized to that recorded at −60 mV. FIGS. 4A and 4B aregraphs illustrating a dose-dependent blockade of ASIC currents byamiloride. IC₅₀=16.4±4.1 04, N=8. FIGS. 4C and 4D are graphsillustrating a blockade of ASIC currents by PcTX venom. **p<0.01.

ASIC currents in cultured mouse cortical neurons were recorded (see FIG.3). At a holding potential of −60 mV, a rapid reduction of extracellularpH (pH_(e)) to below 7.0 evoked large transient inward currents with asmall steady-state component in the majority of neurons (FIG. 3A). Theamplitude of inward current increased in a sigmoidal fashion as pHedecreased, yielding a pH_(0.5) of 6.18±0.06 (n=10, FIG. 3B). A linearI-V relationship and a reversal close to the Na⁺ equilibrium potentialwere obtained (n=6, FIGS. 3C and 3D). These data demonstrate thatlowering pH_(e) may activate typical ASICs in mouse cortical neurons.

The effect of amiloride, a nonspecific inhibitor of ASICs, on theacid-activated currents was tested (see FIG. 4). As shown in FIG. 4,amiloride dose-dependently blocked ASIC currents in cortical neuronswith an IC₅₀ of 16.4±4.1 04 (n=8, FIGS. 4A and 4B). The effect of PcTXvenom on acid-activated current in cortical neurons is shown in FIGS. 4Cand 4D. At 100 ng/mL, PcTX venom reversibly blocked the peak amplitudeof ASIC current by 47%±7% (n=15, FIGS. 4C and 4D), indicatingsignificant contributions of homomeric ASIC1a to total acid-activatedcurrents. Increasing PcTX concentration did not induce further reductionin the amplitude of ASIC current in the majority of cortical neurons(n=8, data not shown), indicating coexistence of PcTX-insensitive ASICs(e.g., heteromeric ASIC1a/2a) in these neurons.

(b) ASIC Response is Potentiated by Modeled Ischemia

FIG. 5 shows exemplary data indicating that modeled ischemia may enhanceactivity of ASICs. FIG. 5A is a series of exemplary traces showing anincrease in amplitude and a decrease in desensitization of ASIC currentsfollowing 1 hr OGD. FIG. 5B is a graph of summary data illustrating anincrease of ASIC current amplitude in OGD neurons. N=40 and 44, *p<0.05.FIG. 5C is a series of exemplary traces and summary data showingdecreased ASIC current desensitization in OGD neurons. N=6, **p<0.01.FIG. 5D is a pair of exemplary traces showing lack of acid-activatedcurrent at pH 6.0 in ASIC1^(−/−) neurons, in control condition, andfollowing 1 hr OGD (n=12 and 13).

Since acidosis may be a central feature of brain ischemia, it wasdetermined to test whether ASICs may be activated in ischemic conditionsand whether ischemia may modify the properties of these channels; seeFIG. 5. ASIC currents in neurons following 1 hr oxygen glucosedeprivation (OGD) were recorded. Briefly, one set of cultures was washedthree times with glucose-free extracellular fluid (ECF) and subjected toOGD, while control cultures were subjected to washes with glucosecontaining ECF and incubation in a conventional cell culture incubator.OGD was terminated after 1 hr by replacing glucose-free ECF withNeurobasal medium and incubating cultures in the conventional incubator.ASIC current was then recorded 1 hr following the OGD when there was nomorphological alteration of neurons. OGD treatment induced a moderateincrease of the amplitude of ASIC currents (1520±138 pA in controlgroup, N=44; 1886±185 pA in neurons following 1 hr OGD, N=40, p<0.05,FIGS. 5A and 5B). More importantly, OGD induced a dramatic decrease inASIC desensitization as demonstrated by an increase in time constant ofthe current decay (814.7±58.9 ms in control neurons, N=6; 1928.9±315.7ms in neurons following OGD, N=6, p<0.01, FIGS. 5A and 5C). In corticalneurons cultured from ASIC1^(−/−) mice, reduction of pH from 7.4 to 6.0did not activate any inward current (n=52), similar to a previous studyin hippocampal neurons (Wemmie et al., 2002). In these neurons, 1 hr OGDdid not activate or potentiate acid-induced responses (FIG. 5D, n=12 and13).

(c) Acidosis Induces Glutamate-Independent Ca²⁺ Entry Via ASIC1a

FIGS. 6 and 7 show exemplary data suggesting that ASICs in corticalneurons may be Ca²⁺ permeable, and that Ca²⁺ permeability may be ASIC1adependent. FIG. 6A shows exemplary traces obtained with Natfree ECFcontaining 10 mM Ca²⁺ as the only charge carrier. Inward currents wererecorded at pH 6.0. The average reversal potential is ˜−17 mV aftercorrection of liquid junction potential (n=5). FIG. 6B showsrepresentative traces and summary data illustrating blockade ofCa²⁺-mediated current by amiloride and PcTX venom. The peak amplitude ofCa²⁺-mediated current decreased to 26%±2% of control value by 100 μMamiloride (n=6, p<0.01) and to 22%±0.9% by 100 ng/mL PcTX venom (n=5,p<0.01). FIG. 7A shows exemplary 340/380 nm ratios as a function of pH,illustrating an increase of [Ca²⁺]_(i) by pH drop to 6.0. Neurons werebathed in normal ECF containing 1.3 mM CaCl₂) with inhibitors forvoltage-gated Ca²⁺ channels (5 μM nimodipine and 1 μM ω-conotoxin MVIIC)and glutamate receptors (10 μM MK801 and 20 μM CNQX). The inset of FIG.7A shows exemplary inhibition of acid-induced increase of [Ca²⁺]_(i) by100 μM amiloride. FIG. 7B shows exemplary summary data illustratinginhibition of acid-induced increase of [Ca²⁺]_(i) by amiloride and PcTXvenom. N=6-8, **p<0.01 compared with pH 6.0 group. FIG. 7C showsexemplary 340/380 nm ratios as a function of pH and NMDApresence/absence, demonstrating a lack of acid-induced increase of[Ca²⁺]_(i) in ASIC1^(−/−) neurons; neurons had a normal response to NMDA(n=8). FIG. 7D shows exemplary traces illustrating a lack ofacid-activated current at pH 6.0 in ASIC1^(−/−) neurons.

The Ca²⁺ permeability of ASICs in cortical neurons was determined usinga standard ion-substitution protocol (Jia et al., Neuron, 1996,17:945-956) and the Fura-2 fluorescent Ca²⁺-imaging technique (Chu etal., 2002, J. Neurophysiol. 87:2555-2561). With bath solutionscontaining 10 mM Ca²⁺ (Na⁺ and K⁺-free) as the only charge carrier andat a holding potential of −60 mV, we recorded inward currents largerthan 50 pA in 15 out of 18 neurons, indicating significant Ca²⁺permeability of ASICs in the majority of cortical neurons (FIG. 6A).Consistent with activation of homomeric ASIC1a channels, currentscarried by 10 mM Ca²⁺ were largely blocked by both the nonspecific ASICinhibitor amiloride and the ASIC1a-specific inhibitor, PcTX venom (FIG.6B). The peak amplitude of Ca²⁺-mediated current was decreased to 26%±2%of control by 100 μM amiloride (n=6, p<0.01) and to 22%±0.9% by 100ng/mL PcTX venom (n=5, p<0.01). Ca²⁺ imaging, in the presence ofinhibitors of other major Ca²⁺ entry pathways (MK801 10 μM and CNQX 20μM for glutamate receptors; nimodipine 5 μM and w-conotoxin MVIIC 1 μMfor voltage-gated Ca²⁺ channels), demonstrated that 18 out of 20 neuronsresponded to a pH drop with detectable increases in the concentration ofintracellular Ca²⁺ ([Ca²⁺]_(i)) (FIG. 7A). In general, [Ca²⁺]_(i)remains elevated during prolonged perfusion of low pH solutions. In somecells, the [Ca²⁺]_(i) increase lasted even longer than the duration ofacid perfusion (FIG. 7A). Long-lasting Ca²⁺ responses suggest that ASICresponse in intact neurons may be less desensitized than in whole-cellrecordings or that Ca²⁺ entry through ASICs may induce subsequent Ca²⁺release from intracellular stores. Preincubation of neurons with 1 μMthapsigargin partially inhibited the sustained component of Ca²⁺increase, suggesting that Ca²⁺ release from intracellular stores mayalso contribute to acid-induced intracellular Ca²⁺ accumulation (n=6,data not shown). Similar to the current carried by Ca²⁺ ions (FIG. 6B),both peak and sustained increases in [Ca²⁺]_(i) were largely inhibitedby amiloride and PcTX venom (FIGS. 7A and 7B, n=6-8), consistent withinvolvement of homomeric ASIC1a in acid-induced [Ca²⁺]_(i) increase.Knockout of the ASICI gene eliminated the acid-induced [Ca²⁺]_(i)increase in all neurons without affecting NMDA receptor-mediated Ca²⁺response (FIG. 7C, n=8). Patch-clamp recordings demonstrated lack ofacid-activated currents at pH 6.0 in 52 out of 52 of the ASIC1^(−/−)neurons, consistent with absence of ASIC1a subunits. Lowering pH to 5.0or 4.0, however, activated detectable current in 24 out of 52ASIC1^(−/−) neurons, indicating the presence of ASIC2a subunits in theseneurons (FIG. 7D). Further electrophysiological studies demonstratedthat ASIC1^(−/−) neurons have normal responses for various voltage-gatedchannels and NMDA, GABA receptor-gated channels (data not shown).

(d) ASIC Blockade Protects Acidosis-Induced, Glutamate-IndependentNeuronal Injury

FIG. 8 shows exemplary data suggesting that acid incubation may induceglutamate receptor-independent neuronal injury protected by ASICblockade. FIGS. 8A and 8B show graphs presenting exemplary data fortime-dependent LDH release induced by 1 hr (FIG. 8A) or 24 hr incubation(FIG. 8B) of cortical neurons in pH 7.4 (solid bars) or 6.0 ECF (openbars). N=20-25 wells, *p<0.05, and **p<0.01, compared to pH 7.4 group atthe same time points (acid-induced neuronal injury with fluoresceindiacetate (FDA) also was analyzed by staining of cell bodies of aliveneurons and propidium iodide (PI) staining of nuclei of dead neurons).FIG. 8C shows a graph illustrating inhibition of acid-induced LDHrelease by 100 μM amiloride or 100 ng/mL PcTX venom (n=20-27, *p<0.05,and **p<0.01). MK801, CNQX, and nimodipine were present in ECF for allexperiments (FIGS. 8A-C).

Acid-induced injury was studied on neurons grown on 24-well platesincubated in either pH 7.4 or 6.0 ECF containing MK801, CNQX, andnimodipine; see FIG. 8. Cell injury was assayed by the measurement oflactate dehydrogenase (LDH) release (Koh and Choi, J. Neurosci., 1987,20:83-90) at various time points (FIGS. 8A and 8B) and by fluorescentstaining of alive/dead cells. Compared to neurons treated at pH 7.4, 1hr acid incubation (pH 6.0) induced a time-dependent increase in LDHrelease (FIG. 8A). After 24 hr, 45.7%±5.4% of maximal LDH release wasinduced (n=25 wells). Continuous treatment at pH 6.0 induced greatercell injury (FIG. 8B, n=20). Consistent with the LDH assay, alive/deadstaining with fluorescein diacetate and propidium iodide showed similarincreases in cell death by 1 hr acid treatment (data not shown). Onehour incubation with pH 6.5 ECF also induced significant but less LDHrelease than with pH 6.0 ECF (n=8 wells, data not shown).

The effects of amiloride and PcTX venom on acid-induced LDH release weretested to determine whether activation of ASICs is involved inacid-induced glutamate receptor-independent neuronal injury. Addition ofeither 100 μM amiloride or 100 ng/mL PcTX venom 10 min before and duringthe 1 hr acid incubation significantly reduced LDH release (FIG. 8C). At24 hr, LDH release was decreased from 45.3%±3.8% to 31.1%±2.5% byamiloride and to 27.9%±2.6% by PcTX venom (n=20-27, p<0.01). Addition ofamiloride or PcTX venom in pH 7.4 ECF for 1 hr did not affect baselineLDH release, although prolonged incubation (e.g., 5 hr) with amiloridealone increased LDH release (n=8, data not shown).

(e) Activation of Homomeric ASIC1a is Responsible for Acidosis-InducedInjury

FIG. 9 is a series of graphs presenting exemplary data indicating thatASIC1a may be involved in acid-induced injury in vitro. FIG. 9A showsexemplary data illustrating inhibition of acid-induced LDH release byreducing [Ca²⁺]_(e) (n=11-12, **p<0.01 compared with pH 6.0, 1.3 Ca²⁺).FIG. 9B shows exemplary data illustrating acid incubation inducedincrease of LDH release in ASIC1a-transfected but not nontransfectedCOS-7 cells (n=8-20). Amiloride (100 μM) inhibited acid-induced LDHrelease in ASIC1a-transfected cells. *p<0.05 for 7.4 versus 6.0 and 6.0versus 6.0+amiloride. FIG. 9C shows exemplary data illustrating a lackof acid-induced injury and protection by amiloride and PcTX venom inASIC1^(−/−) neurons (n=8 in each group, p>0.05). FIG. 9D shows exemplarydata illustrating acid-induced increase of LDH release in culturedcortical neurons under OGD (n=5). LDH release induced by combined 1 hrOGD/acidosis was not inhibited by trolox and L-NAME (n=8-11). OGD didnot potentiate acid-induced LDH release in ASIC1^(−/−) neurons. **p<0.01for pH 7.4 versus pH 6.0 and *p<0.05 for pH 6.0 versus 6.0+PcTX venom.MK801, CNQX, and nimodipine were present in ECF for all experiments(FIG. 9A-D).

Neurons were treated with pH 6.0 ECF in the presence of normal orreduced [Ca²⁺]_(e) to determine whether Ca²⁺ entry plays a role inacid-induced injury (see FIG. 9). Reducing Ca²⁺ from 1.3 to 0.2 mMinhibited acid-induced LDH release (from 40.0%±4.1% to 21.9%±2.5%), asdid ASIC1a blockade with PcTX venom (n=11-12, p<0.01; FIG. 9A). Ca²⁺-free solution was not tested, as a complete removal of [Ca²⁺]_(e) mayactivate large inward currents through a Ca²⁺-sensing cation channel,which may otherwise complicate data interpretation. Inhibition of acidinjury by both amiloride and PcTX, nonspecific and specific ASIC1ainhibitors, and by reducing [Ca²⁺]_(e) suggests that activation ofCa²⁺-permeable ASIC1a may be involved in acid-induced neuronal injury.

Acid injury of nontransfected and ASIC1a transfected COS-7 cells wasstudied to provide additional evidence that activation of ASIC1a isinvolved in acid injury. COS-7 is a cell line commonly used forexpression of ASICs due to its lack of endogenous channels. Followingconfluence (36-48 hr after plating), cells were treated with either pH7.4 or 6.0 ECF for 1 hr. LDH release was measured 24 hr after acidincubation. Treatment of nontransfected COS-7 cells with pH 6.0 ECF didnot induce increased LDH release when compared with pH 7.4-treated cells(10.3%±0.8% for pH 7.4, and 9.4%±0.7% for pH 6.0, N=19 and 20 wells;p>0.05, FIG. 9B). However, in COS-7 cells stably transfected withASIC1a, 1 hr incubation at pH 6.0 significantly increased LDH releasefrom 15.5%±2.4% to 24.0%±2.9% (n=8 wells, p<0.05). Addition of amiloride(100 μM) inhibited acid-induced LDH release in these cells (FIG. 9B).

Acid injury of CHO cells transiently transfected with cDNAs encoding GFPalone or GFP plus ASIC1a was also studied. After the transfection (24-36hr), cells were incubated with acidic solution (pH 6.0) for 1 hr, andcell injury was assayed 24 hr following the acid incubation. One houracid incubation largely reduced surviving GFP-positive cells inGFP/ASIC1a group but not in the group transfected with GFP alone (datanot shown).

Cell toxicity experiments on cortical neurons cultured from ASIC^(+/+)and ASIC1^(−/−) mice were performed to further demonstrate aninvolvement of ASIC1a in acidosis-induced neuronal injury. Again, 1 hracid incubation of ASIC^(+/+) neurons at 6.0 induced substantial LDHrelease that was reduced by amiloride and PcTX venom (n=8-12). One houracid treatment of ASIC1^(−/−) neurons, however, did not inducesignificant increase in LDH release at 24 hr (13.8%±0.9% for pH 7.4 and14.2%±1.3% for pH 6.0, N=8, p>0.05), indicating resistance of theseneurons to acid injury (FIG. 9C). In addition, knockout of the ASIC1gene also eliminated the effect of amiloride and PcTX venom onacid-induced LDH release (FIG. 9C, n=8 each), further suggesting thatthe inhibition of acid-induced injury of cortical neurons by amilorideand PcTX venom (FIG. 8C) was due to blockade of ASIC1 subunits. Incontrast to acid incubation, 1 hr treatment of ASIC1^(−/−) neurons with1 mM NMDA+10 μM glycine (in Mg²⁺-free [pH 7.4]ECF) induced 84.8%±1.4% ofmaximal LDH release at 24 hr (n=4, FIG. 9C), indicating normal responseto other cell injury processes.

(f) Modeled Ischemia Enhances Acidosis-Induced Glutamate-IndependentNeuronal Injury Via ASICs

As the magnitude of ASIC currents may be potentiated by cellular andneurochemical components of brain ischemia-cell swelling, arachidonicacid, and lactate and, more importantly, the desensitization of ASICcurrents may be reduced dramatically by modeled ischemia (see FIGS. 5Aand 5C), activation of ASICs in ischemic conditions is expected toproduce greater neuronal injury. To test this hypothesis, neurons weresubjected to 1 hr acid treatment under oxygen and glucose deprivation(OGD). MK801, CNQX, and nimodipine were added to all solutions toinhibit voltage-gated Ca²⁺ channels and glutamate receptor-mediated cellinjury associated with OGD. One hour incubation with pH 7.4 ECF underOGD conditions induced only 27.1%±3.5% of maximal LDH release at 24 hr(n=5, FIG. 9D). This finding is in agreement with a previous report that1 hr OGD does not induce substantial cell injury with the blockade ofglutamate receptors and voltage-gated Ca²⁺ channels (Aarts et al.,2003). However, 1 hr OGD, combined with acidosis (pH 6.0), induced73.9%±4.3% of maximal LDH release (n=5, FIG. 9D, p<0.01), significantlylarger than acid-induced LDH release in the absence of OGD (see FIG. 8A,p<0.05). Addition of the ASIC1a inhibitor PcTX venom (100 ng/mL)significantly reduced acid/OGD-induced LDH release to 44.3%±5.3% (n=5,p<0.05, FIG. 9D).

The same experiment was performed with cultured neurons from theASIC1^(−/−) mice. Unlike in ASICI containing neurons, however, 1 hrtreatment with combined OGD and acid only slightly increased LDH releasein ASIC1^(−/−) neurons (from 26.1%±2.7% to 30.4%±3.5%, N=10-12, FIG.9D). This finding suggests that potentiation of acid-induced injury byOGD may be due largely to OGD potentiation of ASIC1-mediated toxicity.

It has been demonstrated that activation of a Ca²⁺-permeablenonselective cation conductance activated by reactive oxygen/nitrogenspecies resulting in glutamate receptor-independent neuronal injury(Aarts et al, Cell, 2003, 115:863-877). The prolonged OGD-induced cellinjury may be reduced dramatically by agents either scavenging freeradicals directly (e.g., trolox) or reducing the production of freeradicals (e.g., L-NAME). To determine whether combined short durationOGD and acidosis induced neuronal injury may involve a similarmechanism, the effect of trolox and L-NAME on OGD/acid-induced LDHrelease was tested. As shown in FIG. 9D, neither trolox (500 μM) norL-NAME (300 μM) had significant effect on combined 1 hrOGD/acidosis-induced neuronal injury (n=8-11). Additional experimentsalso demonstrated that the ASIC inhibitors amiloride and PcTX venom hadno effect on the conductance of TRPM7 channels (Aarts et al. supra).Together, these findings strongly suggest that activation of ASICs butnot TRPM7 channels may be largely responsible for combined 1 hrOGD/acidosis-induced neuronal injury in our studies.

(g) Activation of ASIC1a in Ischemic Brain Injury In Vivo

FIG. 10 shows data illustrating neuroprotection by ASICI blockade andASICI gene knockout in brain ischemia in vivo. FIG. 10A shows a graph ofexemplary data obtained from TTC-stained brain sections illustrating thestained volume (“infarct volume”) in brains from aCSF (n=7), amiloride(n=11), or PcTX venom (n=5) injected rats. *p<0.05 and **p<0.01 comparedwith aCSF injected group. FIG. 10B shows a graph of exemplary dataillustrating reduction in infarct volume in brains from ASIC1^(−/−) mice(n=6 for each group). *p<0.05 and **p<0.01 compared with +/+ group. FIG.10C shows a graph of exemplary data illustrating reduction in infarctvolume in brains from mice i.p. injected with 10 mg/kg memantine (Mem)or i.p. injection of memantine accompanied by i.c.v. injection of PcTXvenom (500 ng/mL). **p<0.01 compared with aCSF injection and betweenmemantine and memantine plus PcTX venom (n=5 in each group). FIG. 10Dshows a graph of exemplary data illustrating reduction in infarct volumein brains from either ASIC1^(+/+) (wt) or ASIC1^(−/−) mice i.p. injectedwith memantine (n=5 in each group). *p<0.05, and **p<0.01.

The protective effect of amiloride and PcTX venom in a rat model oftransient focal ischemia (Longa et al., Stroke, 1989, 20:84-91) wastested to determine whether activation of ASIC1a is involved in ischemicbrain injury in vivo. Ischemia (100 min) was induced by transient middlecerebral artery occlusion (MCAO). A total of 6 μl artificial CSF (aCSF)alone, aCSF-containing amiloride (1 mM), or PcTX venom (500 ng/mL) wasinjected intracerebroventricularly 30 min before and after the ischemia.The volume for cerebral ventricular and spinal cord fluid for 4-week-oldrats is estimated to be ˜60 μl. Assuming that the infused amiloride andPcTX were uniformly distributed in the CSF, a concentration of ˜100 μMfor amiloride and ˜50 ng/mL for PcTX were expected, which is aconcentration found effective in cell culture experiments. Infarctvolume was determined by TTC staining (Bederson et al., Stroke, 1986,17:1304-1308) at 24 hr following ischemia. Ischemia (100 min) producedan infarct volume of 329.5±25.6 mm³ in aCSF-injected rats (n=7) but only229.7±41.1 mm³ in amiloride-injected (n=11, p<0.05) and 130.4±55.0 mm³(˜60% reduction) in PcTX venom-injected rats (n=5, p<0.01) (FIG. 10A).

ASIC1^(−/−) mice were used to further demonstrate the involvement ofASIC1a in ischemic brain injury in vivo. Male ASIC1^(+/+), ASIC1^(−/−),and ASIC1^(−/−) mice (˜25 g, with congenic C57B16 background) weresubjected to 60 min MCAO as previously described (Stenzel-Poore et al.,Lancet, 2003, 362:1028-1037). Consistent with the protection bypharmacological blockade of ASIC1a (above), −/− mice displayedsignificantly smaller (˜61% reduction) infarct volumes (32.9±4.7 mm³,N=6) as compared to +/+ mice (84.6±10.6 mm³, N=6, p<0.01). +/− mice alsoshowed reduced infarct volume (56.9+−0.6.7 mm³, N=6, p<0.05) (FIG. 10B).

To determine whether blockade of ASIC1a channels or knockout of theASIC1 gene would provide additional protection in vivo in the setting ofglutamate receptor blockade, memantine (10 mg/kg) was injectedintraperitoneally (i.p.) into C57B16 mice immediately following 60 minMCAO and accompanied by intracerebroventricular injection (i. c. v.) ofa total volume of 0.4 μl aCSF alone or aCSF containing PcTX venom (500ng/mL) 15 min before and following ischemia. In control mice with i.p.injection of saline and i.c.v. injection of aCSF, 60 min MCAO induced aninfarct volume of 123.6±5.3 mm³ (n=5, FIG. 10C). In mice withintraperitoneal injection of memantine and intracerebroventricularinjection of aCSF, the same duration of ischemia induced an infarctvolume of 73.8+−0.6.9 mm³ (n=5, p<0.01). However, in mice injected withmemantine and PcTX venom, an infarct volume of only 47.0±1.1 mm³ wasinduced (n=5, p<0.01 compared with both control and memantine groups,FIG. 10C). These data suggest that blockade of homomeric ASIC1a mayprovide additional protection in in vivo ischemia in the setting of NMDAreceptor blockade. Additional protection was also observed inASIC1^(−/−) mice treated with pharmacologic NMDA blockade (FIG. 10D). InASIC^(+/+) mice i.p. injected with saline or 10 mg/kg memantine, 60 minMCAO induced an infarct volume of 101.4±9.4 mm³ or 61.6±12.7 mm³,respectively (n=5 in each group, FIG. 10D). However, in ASIC1^(−/−)miceinjected with memantine, the same ischemia duration induced an infarctvolume of 27.7±1.6 mm³ (n=5), significantly smaller than the infarctvolume in ASIC1^(+/+) mice injected with memantine (p<0.05).

Taken together, these data demonstrate that activation of Ca²⁺-permeableASIC1a is a novel, glutamate-independent biological mechanism underlyingischemic brain injury.

Example 2: Time Window of PcTX Neuroprotection

This example describes exemplary experiments that measure theneuroprotective effect of PcTX venom at different times after onset ofstroke in rodents; see FIG. 11. Briefly, brain ischemia (stroke) wasinduced in rodents by mid-cerebral artery occlusion (MCAO). At theindicated times after induction, artificial cerebrospinal fluid (aCSF),PcTX venom (0.5 μL, 500 ng/mL total protein), or inactivated (boiled)venom was infused into the lateral ventricles of each rodent. As shownin FIG. 11, administration of PcTX venom provided a 60% reduction instroke volume both at one hour and at three hours after stroke onset.Furthermore, substantial stroke volume reduction still may be maintainedif treatment is withheld for five hours after the onset of the MCAO.Accordingly, neuroprotection due to ASIC inhibition may have an extendedtherapeutic time window after stroke onset, allowing stroke subjects tobenefit from treatment performed hours after the stroke began. Thiseffect of ASIC blockade on stroke neuroprotection is far more robustthan that of calcium channel blockade of the NMDA receptor (a majortarget for experimental stroke therapeutics) using a glutamateantagonist. No glutamate antagonist, thus far, has such a favorableprofile as shown here for ASIC 1a-selective inhibition.

Example 3: Exemplary Cystine Knot Peptides

This example describes exemplary cystine knot peptides, includingfull-length PcTx1 and deletion derivatives of PcTx, which may bescreened in cultured cells, tested in ischemic animals (e.g., rodentssuch as mice or rats), and/or administered to ischemic human subjects.

FIG. 12 shows the primary amino acid sequence (SEQ ID NO:1), inone-letter code, of an exemplary cystine knot peptide, PcTx1, indicatedat 50, with various exemplary peptide features shown relative to aminoacid positions 1-40. Peptide 50 may include six cysteine residues thatform cystine bonds 52, 54, 56 to create a cystine knot motif 58. Thepeptide also may include one or more beta sheet regions 60 and apositively charged region 62. An N-terminal region 64 and a C-terminalregion 66 may flank the cystine knot motif.

FIG. 13 shows a comparison of the PcTx1 peptide 50 of FIG. 12 alignedwith various exemplary deletion derivatives of the peptide. Thesederivatives may include an N-terminal deletion 70 (SEQ ID NO:2), apartial C-terminal deletion 72 (SEQ ID NO:3), a full C-terminal deletion74 (SEQ ID NO:4), and an N/C terminal deletion 76 (SEQ ID NO:5). Otherderivatives of PcTx1 may include any deletion, insertion, orsubstitution of one or more amino acids, for example, while maintainingsequence similarity or identity of at least about 25% or about 50% withthe original PcTx1 sequence.

Each PcTx1 derivative may be tested for its ability to inhibit ASICproteins selectively and/or for an effect, if any, on ischemia. Anysuitable test system(s) may be used to perform this testing includingany of the cell-based assay systems and/or animal model systemsdescribed elsewhere in the present teachings. The PcTx1 derivative alsoor alternatively may be tested in ischemic human subjects.

Example 4: Selectivity of PcTX Venom for ASIC1a

This example describes experiments that measure the selectivity of PcTXvenom (and thus PcTx1 toxin) for ASIC1a alone, relative to other ASICproteins or combinations of ASIC proteins expressed in cultured cells.COS-7 cells expressing the indicated ASIC proteins were treated withPcTX venom (25 ng/mL on ASIC1a expressing cells and 500 ng/mL on ASIC2a,ASIC3 or ASIC1a+2a expressing cells). Channel currents were measured atthe pH of half maximal channel activation (pH 0.5). As shown in FIG. 14,PcTX venom largely blocked the currents mediated by ASIC1a homomericchannels at a protein concentration of 25 ng/mL, with no effect on thecurrents mediated by homomeric ASIC2a, ASIC3, or heteromericASIC1a/ASIC2a at 500 ng/mL (n=3-6). At 500 ng/mL, PcTX venom also didnot affect the currents mediated by other ligand-gated channels (e.g.,NMDA and GABA receptor-gated channels) and voltage-gated channels (e.g.,Na+, Ca2+, and K+ channels) (n=4-5). These experiments indicate thatPcTX venom and thus PcTx1 peptide is a specific inhibitor for homomericASIC1a. Using this cell-based assay system, the potency and selectivityof ASIC inhibition may be measured for various synthetic peptides orother candidate inhibitors (e.g., see Example 3).

Example 5: Nasal Administration of PcTX Venom is Neuroprotective

This example describes exemplary data indicating the efficacy of nasallyadministered PcTX venom for reducing ischemia-induced injury in ananimal model system of stroke. Cerebral ischemia was induced in malemice by mid-cerebral artery occlusion. One hour after occlusion wasinitiated animals were treated as controls or were treated with PcTXvenom (50 μL of 5 ng/mL (total protein) PcTx venom introducedintranasally). As shown in FIG. 15, nasal administration of PcTX venomresulted in a 55% reduction in ischemia-induced injury (ischemicdamage), as defined by infarct volume, relative to control treatment.Nasal administration may be via a spray that is deposited substantiallyin the nasal passages rather than inhaled into the lungs and/or may bevia an aerosol that is at least partially inhaled into the lungs. Insome examples, nasal administration may have a number of advantages overother routes of administration, such as more efficient delivery to thebrain and/or adaptability for self-administration by an ischemicsubject.

Example 6: Inhibition of ASIC1a Channel by Amiloride and AmilorideAnalogs

As shown in FIG. 16, amiloride and amiloride analogs benzamil, phenamiland EIPA block ASIC1a current in a dose-dependent manner. Similarly,amiloride and amiloride analogs benzamil and EIPA block ASIC2a currentin a dose-dependent manner (FIG. 17). Table 1 summarizes inhibition ofthe ASIC1a channel by amiloride and amiloride analogs. Amiloride was aneffective inhibitor of this channel with an IC50 of 7.7 μM.

TABLE 1 Inhibition of the ASIC1a channel by amiloride and amilorideanalogs. Knockout 1a 39.1 ± 3.8 (n = 4)  Knockout 2a 5.14 ± 0.79 (n = 5)Neuron (−60 mV) 43.3 ± 1.4 (n = 6)  EIPA Neuron (−20 mV) 32.2 ± 6.3 (n =3)  CHO 1a 111 ± 30 (n = 5)  CHO 2a 31 (n = 1) Knockout 1a 35.9 ± 2.1 (n= 5)  Knockout 2a 20.1 ± 2.2 (n = 2)  Neuron (−60 mV) 82.9 ± 5.2 (n =8)  Bepridil Neuron (−60 mV) 100 ± 11 (n = 10) KB-R7943 Neuron (−60 mV)24.3 ± 17.2 (n = 2) 5-(N-methyl-N-isobutyl) amiloride Neuron (−60 mV)15.0 ± 11.7 (n = 3) 5-(N,N-hexamethylene) amiloride Neuron (−60 mV) 14.8± 7.1 (n = 2)  5-(N,N-dimethyl) amiloride hydrochloride

Example 7: Reduction of Infarct Volume in Mice byIntracerebroventricular Injection of Amiloride and Amiloride Analogs

Mice were subjected to 60 minutes of middle cerebral artery occlusion(MCAO) as described above. Amiloride or an amiloride analog benzamil,bepridil, EIPA or KB-R7943 was administered by intracerebroventricularinjection one hour after MCAO. The animals were evaluated one day afterischemia induction. As shown in FIG. 18, intracerebroventricularinjection of amiloride or an amiloride analog benzamil, bepridil, EIPAor KB-R7943 effectively reduce infarct volume.

Example 8: Reduction of Infarct Volume in Mice by Intravenous Injectionof Amiloride

Mice were subjected to 60 minutes of middle cerebral artery occlusion(MCAO) as described above. Amiloride was administered by intravenousinjection 1, 3 or 5 hours after MCAO. The animals were evaluated one dayafter ischemia induction. As shown in FIG. 19, intravenous injection ofamiloride effectively reduces infarct volume. The effective CNSpenetration of amiloride may be explained by the fact thatblood-brain-barrier is compromised following brain ischemia/reperfusion.FIG. 20 shows that intravenous injection of amiloride has a prolongedtherapeutic window of 5 h.

Example 9: Structure Activity Relationships for Hydrophobic AmilorideAnalogs on Various Channels

As shown in Table 1, substituting the C-5 amino group in amiloride withalkyl groups led to a decrease in potency at the ASIC1a channel. Thesame substitution increases potency to the ASIC3 channel (Kuduk et al.,Bioorg. Med. Chem. Lett., 2009, 19:2514-2518). The reverse result wasobtained when substituting hydrophobic groups onto the guanidino part ofthe structure. Indeed, the benzyl substituted guanidino analog,benzamil, was the most potent ASIC1a blocking compound tested (IC₅₀=4.9μM). Taken together, these results showed that amiloride is an effectiveinhibitor of ASIC1a with an IC₅₀ of 7.7 μM. They also provide structureactivity relationships (see FIG. 21) for designing amiloride analogsthat would inhibit the ASIC1a channel. Accordingly, in some embodiments,amiloride analogs are generated by introducing changes in the guanidineportion of the amiloride structure. Since amiloride is only a very weakinhibitor of the Na⁺/Ca²⁺ ion exchanger (IC₅₀=1.1 mM). The amilorideanalogs are likely be a very weak inhibitor of the Na⁺/Ca²⁺ ionexchanger as well. In some embodiments, the amiloride analogs aredesigned to have increased selectivity for ASIC1a over the ASIC3channel. In other embodiments, a ring structure, such as a cyclicguanidine group, is introduced into the amiloride structure to increaseinhibitory potency of ASIC1a currents. It is also possible that one ormore of the N—H groups of amiloride will form H-bonds either internallywith the 3-amino group or with the ion channel.

The in vivo results in mice showed that efficacy can be achieved with aplasma concentration of 32.5 μM (iv dose of 50 μl×1 mM) and a totalbrain concentration of 12.5 μM (icy dose of 1 μl×500 μM). It is thusestimated that only a 10-fold increase in potency is necessary toachieve efficacious concentrations that are suitable for an acutetherapeutic for stroke in humans. Therefore, novel analogs are screenedfor an increased ASIC1a IC₅₀ potency from the 4 to 8 μM for amilorideand benzamil to <1 μM.

In some embodiments, the amiloride analogs comprise methylated analogsof benzamil (formula 1-5 of FIG. 21) and amidino analog of benzamil(formula 6 of FIG. 21). In other embodiments, the amiloride analogscontain a ring formed on the guanidine group. In other embodiments, theamiloride analogs contain an acylguanidino group for increasedinhibitory potency of ASIC1a currents.

Amiloride is soluble in water at 1 mM and is effective in treatingischemia in a mouse model at a dose of 50 ul per injection. Theequivalent dose on a mg/kg basis in a 65 kg human would be close to 40mg and require an injection volume of over 160 ml. Similarly benzamilhas a reported solubility in 0.9% saline of 0.4 mg/ml (1.7 mM), whichpermits administration of only 5 mg benzamil dihydrochloride in a 10 mlinjection. Accordingly, amiloride analogs with higher water solubilityare desired. In some embodiments, the amiloride analogs contain a watersolubilizing group, such as an N,N-dimethyl amino group or a sugar, atthe guanidino group to improve water solubility. In some embodiments,the amiloride analogs have a water solubility of 5 mM, 10 mM, 20 mM, 30mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or higher. In otherembodiments, the amiloride analogs have a solubility that allows for a10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or500 mg dose to be administered intravenously to a human in a single 10ml injection. In yet other embodiments, the amiloride analogs have asolubility that allows for a 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200mg, 250 mg, 300 mg, 400 mg, or 500 mg dose to be administeredintracerebroventicularly to a human in a single 2 ml injection.

Example 10: Use of Amiloride and Amiloride Analogues in TransientIschemic Attack Model

To evaluate the neuroprotective potential of amiloride and its analogs,a transient focal ischemia model in mice (Li et al., Brain Research,1055:180-185, 2005; Pedrono et al., J. Neuropathol Exp. Neurol.,69(2):188-195, 2010) or rats (Tolvanen et al., Brain Research,1663:166-173, 2017) is employed with or without the anti-clotting agentsdescribed herein. Prior to induction of transient focal ischemia,animals are pre-treated for 5-10 days with amiloride, amiloride analogs,and/or anti-clotting agents by gavage or intraperitoneal injection(e.g., 1-60 mg/kg/day each). Middle cerebral artery occlusion (MCAO) isinduced using an intraluminal monofilament. The tip of a surgical suture(6-0 nylon monofilament, Ethicon, UK) is blunted or rounded by heating,introduced into the common carotid artery (CCA) and then advancedintracranially to the origin of the middle cerebral artery (MCA) toblock the blood flow into that artery. After the insertion of themonofilament (thereby the start of the MCA occlusion), the monofilamentwas secured in place for a period of 5-10 min. After that period ofMCAO, the monofilament was withdrawn to allow reperfusion into the MCAfor 24 hours. Under these conditions, no detectable brain infarction isexpected.

The effects of the active agents on % hemispheric lesion volume, brainoedema, rotarod performance, spontaneous locomotor activity andmortality rate are assessed. The NMDA channel blocker and gold standardNMDA antagonist, MK-801 may be used as a control.

Regional cerebral blood flow (CBF) is measured in animals by laserDoppler flowmetry (LDF) by applying a flexible fiber-optic probe ontothe intact and bare cranial surface on the territory receiving bloodsupply from the MCA (1 mm posterior and 3 mm lateral to the bregma).Cerebral blood flow values at baseline, occlusion, and reperfusion(immediately, and at 10, 20, 30, and 24 hours after reperfusion) arerecorded.

At the end of the reperfusion period (i.e., at 24 hours), the functionalconsequences of the transient ischemic insult are evaluated using a5-point scale of neurological status (or sensorimotor skills) asfollows: 0, no deficit; 1, failure to fully extend the left or rightpaw; 2, circling to the left or right; 3, decreased resistance tolateral push; 4, unable to walk spontaneously.

Following reperfusion and evaluation of neurological status, the animalsare sacrificed and their brains are harvested, fixed in formaldehyde,and embedded in paraffin blocks. 4-6 μm-thick sections are cut with amicrotome and are subjected to staining with hematoxylin and eosin(H&E), immunohistochemical staining (e.g., fibrinogen, glycoprotein 1beta (GP1b) for detection of thrombus development) and/or apoptosisstaining (e.g., TUNEL kit; (In Situ Cell Death Detection Kit,Fluorescein; Sigma-Aldrich, St. Louis, Mo.). The above-described assaysare used to demonstrate the extent of ischemic changes and to correlatethese changes with the prophylactic benefits conferred by ASIC1ainhibition, with or without anti-clotting agents described in thepresent disclosure.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. A method for preventing or reducing the incidenceof a transient ischemic attack in a subject at risk for developing astroke, comprising: orally administering to the subject aprophylactically effective amount of a pharmaceutical compositioncomprising an ASIC1a inhibitor capable of penetrating the blood-brainbarrier.
 2. The method of claim 1, wherein the ASIC1a inhibitorcomprises amiloride, an amiloride analog, or a pharmaceuticallyacceptable salt thereof.
 3. The method of claim 1, wherein the ASIC1ainhibitor comprises amiloride or a pharmaceutically acceptable saltthereof.
 4. The method of claim 1, wherein the ASIC1a inhibitorcomprises an amiloride analog or a pharmaceutically acceptable saltthereof.
 5. The method of claim 4, wherein the amiloride analog isselected from the group consisting of benzamil, bepridil, KB-R7943,phenamil, 5-(N—N-dimethyl) amiloride (DMA), 5-(N,N-hexamethylene)amiloride (HMA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA),5-(N-methyl-N-isopropyl (MIA), pharmaceutically acceptable salts orsolvates thereof, methylated analogs thereof, and combinations thereof.6. The method of claim 4, wherein the amiloride analog is selected fromthe group consisting of methylated analogs of benzamil, amilorideanalogs containing a ring formed on a guanidine group, amiloride analogscontaining an acylguanidino group, and amiloride analogs containing awater solubilizing group formed on a guanidine group, wherein the watersolubilizing group is a N,N-dimethyl amino group or a sugar group. 7.The method of claim 1, wherein the pharmaceutical composition isadministered daily.
 8. The method of claim 1, wherein the pharmaceuticalcomposition is formulated as an extended release formulation.
 9. Themethod of claim 1, wherein the subject has recently had heart surgery orhas previously had a TIA or stroke.
 10. The method of claim 1, whereinthe subject has an abnormal heart rhythm selected from the groupconsisting of atrial fibrillation, atrial flutter, ventriculartachycardia, and ventricular fibrillation.
 11. The method of claim 1,wherein the subject has acute coronary syndrome, arterial embolism,atherosclerosis, atrial fibrillation, carotid artery disease, cerebralarterial thrombosis, cerebral embolism, coronary arterial thrombosis,coronary heart disease, deep vein thrombosis, kidney embolism,myocardial infarction, peripheral arteriopathy, pulmonary embolism,stroke, thrombophlebitis, thrombosis, transient ischemic attack,unstable angina, valvular heart disease, venous thrombosis, ventricularfibrillation, or a combination thereof.
 12. The method of claim 1,wherein the amiloride, amiloride analog or a pharmaceutically acceptablesalt or solvate thereof is administered in a dose range of 0.1 mg-10mg/kg body weight.
 13. The method of claim 1, further comprising orallyadministering to the subject one or more anti-clotting agents.
 14. Themethod of claim 13, wherein the one or more anti-clotting agentscomprise an antiplatelet agent selected from the group consisting ofaspirin, clopidogrel, prasugrel, ticagrelor, dipyridamole, andcombinations thereof.
 15. The method of claim 13, wherein the one ormore anti-clotting agents comprise an anticoagulant selected from thegroup consisting of vitamin-K epoxide reductase inhibitors, directthrombin inhibitors and direct Factor Xa inhibitors.
 16. The method ofclaim 14, wherein the anticoagulant is selected from the groupconsisting of apixaban, argatroban, AZD-0837, betrixaban, dabigatran,edoxaban, heparin, rivoraxaban, tecarfarin, warfarin, ximelagatran,YM466, and combinations thereof.
 17. The method of claim 1, wherein theone or more anti-clotting agents comprise an anti-arrhythmic agentselected from the group consisting of amiodarone, AZD-1305, budiodarone,celivarone, dofetilide, dronedarone, flecainide, ibutilide, propafenone,quinidine, sotolol, vernakalant, and combinations thereof.
 18. Apharmaceutical composition for reducing nervous system injury,comprising: an effective amount of one or more ASIC1a inhibitorsselected from the group consisting of amiloride, an amiloride analog, apharmaceutically acceptable salt thereof, a methylated analog thereof,and a combination thereof; and a pharmaceutically acceptable carrier,wherein the pharmaceutical composition is formulated for oraladministration of the one or more ASIC1a inhibitor(s), wherein theASIC1a inhibitor(s) are capable of penetrating the blood-brain barrier.19. The pharmaceutical composition of claim 18, wherein the ASIC1ainhibitor comprises amiloride or a pharmaceutically acceptable saltthereof.
 20. The pharmaceutical composition of claim 16, wherein theASIC1a inhibitor comprises an amiloride analog or a pharmaceuticallyacceptable salt thereof.
 21. The pharmaceutical composition of claim 18,wherein the amiloride analog is selected from the group consisting ofbenzamil, bepridil, KB-R7943, phenamil, 5-(N—N-dimethyl) amiloride(DMA), 5-(N,N-hexamethylene) amiloride (HMA),5-(N-ethyl-N-isopropyl)-amiloride (EIPA), 5-(N-methyl-N-isopropyl (MIA),a pharmaceutically acceptable salt thereof, a methylated analog thereof,and a combination thereof.
 22. The pharmaceutical composition of claim18, wherein the amiloride analog is selected from the group consistingof methylated analogs of benzamil, amiloride analogs containing a ringformed on a guanidine group, amiloride analogs containing anacylguanidino group, and amiloride analogs containing a watersolubilizing group formed on a guanidine group, wherein the watersolubilizing group is a N,N-dimethyl amino group or a sugar group 23.The pharmaceutical composition of claim 18, wherein the pharmaceuticalcomposition comprises an extended release formulation for delivery ofthe one or more ASIC1a inhibitors.
 24. The pharmaceutical composition ofclaim 18, further comprising one or more anti-clotting agents.
 25. Thepharmaceutical composition of claim 22, wherein the one or more clottingagents comprise an antiplatelet agent selected from the group consistingof aspirin, clopidogrel, prasugrel, ticagrelor, dipyridamole, andcombinations thereof.
 26. The pharmaceutical composition of claim 22,wherein the one or more clotting agents comprise an anticoagulantselected from the group consisting of apixaban, argatroban, AZD-0837,betrixaban, dabigatran, edoxaban, heparin, rivoraxaban, tecarfarin,warfarin, ximelagatran, YM466, and combinations thereof.
 27. Thepharmaceutical composition of claim 22, wherein the one or more clottingagents comprise an anti-arrhythmic agent selected from the groupconsisting of amiodarone, AZD-1305, budiodarone, celivarone, dofetilide,dronedarone, flecainide, ibutilide, propafenone, quinidine, sotolol,vernakalant, and combinations thereof.